Fluidics device

ABSTRACT

The present invention contemplates various devices that are configured to separate a sample, which contains more than one unique species, into any desired number of sub-samples by passing the sample across a like number of separation media configured for a first separation protocol. Each of the sub-samples may be further separated by an additional separation protocol, thereby creating a plurality of mini-samples, each of which may be further separated and/or analyzed. The invention also contemplates using a simple method of using conduits to form a fluid path that passes through a plurality of separation media, each of which media is configured to isolate a particular sub-sample. After various sub-samples of the sample are isolated by the various separation media, the conduits may be removed, thereby enabling each of the isolated sub-samples to be further separated and/or analyzed independent of any other sub-sample.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 11/915,151, filed Aug. 4, 2009, which is a U.S. National Phaseof PCT Patent Application No. PCT/US2006/001687 filed Jan. 19, 2006, andclaims priority to U.S. Provisional Patent Application No. 60/684,177filed May 25, 2005 and U.S. Provisional Patent Application No.60/702,989 filed Jul. 28, 2005, each of which is incorporated herein byreference in its entirety for all purposes.

BACKGROUND

This application claims the benefit of the priority date of U.S.Provisional Application Ser. Nos. 60/684,177 filed on May 25, 2005(“Fluidics Device,” Boschetti et al.) and 60/702,989, filed Jul. 28,2005 (“Separation Of Proteins Based On Isoelectric Point UsingSolid-Phase Buffers,” Boschetti et al.).

Current materials and methods for isolating the species in a givenbiological sample simply are not sufficient to isolate reliably all ofthe components of such a mixture. Typically, the dominant molecularspecies will mask those species present in concentrations less thanabout one one thousandth of the dominant species. For biologicalsamples, such as blood, two of the most predominant molecular speciesare albumin and immunoglobulins. Attempts to identify various enzymes,antibodies, proteins, or secondary metabolites that may have relevanceas disease markers, or which may be relevant for drug discovery, arecomplicated by the overall high abundance of albumin andimmunoglobulins. As a result, the conventional resolving power,sensitivity, and loading capacity of the two most commonly usedanalytical techniques (i.e., 2-dimensional electrophoresis (2DE) andmass spectrometry) are limited. For example, the presence of such highlyabundant proteins in a sample produces large signals with consequentsignal overlap (in 2DE) or signal suppression (in mass spectrometry) ofthe other species present in the sample, which complicates analysis andundermines any conclusions about the catalog of molecular speciespresent in the sample.

To isolate particular molecules, various separation protocols have beendeveloped. For example, in gel electrophoresis proteins are uniformlycoated with a negatively-charged detergent (e.g. SDS) and placed in themiddleon one end (origin) of a buffered gel (e.g., polyacrylamide gel)between oppositely charged electrodes. When the electrodes are charged,each of the protein molecules travels toward one of the electrodes,according to their net charge at the pH of the buffered polyacrylamidegelthe oppositely charged electrode. The speed, or mobility, at whichthe protein molecules move through the gel toward the electrodes islargely dependent on the size of the molecule, i.e., smaller moleculesmove faster through the gel matrix. As a result in the differences inmobility, types of protein molecules can be separated and then isolatedbased on their size.

A variant to gel electrophoresis is isoelectric focusing, which exploitsthe fact that the net charge of a protein depends on the environmentalpH. Most generally, at acidic pH, proteins are globally positivelycharged while in alkaline pH they are negatively charged. The pH atwhich a protein has no net charge is called its isoelectric point(“pI”). Isoelectric focusing is an electrophoresis technique in whichproteins move under an electric field through a pH gradient. Allproteins migrate towards the cathode or the anode until they encounter apH identical to their isoelectric point. At this isoelectric point theprotein loses its charge and stops moving.

Proteins of different isoelectric points stop at differentlevelspositions and are thus separated for subsequent identification.Accordingly, similarly sized molecules, which may move at similarspeeds, can be separated after coming to rest at different pH points, asa result of having different pI values. In addition, there aresituations in which migration by the size in a given buffered gel andmigration by the isoelectric point are crossed for an enhancedseparation of protein species from very complex mixtures; the techniqueused in this situation is called bidimensional electrophoresis.Unfortunately, migration of proteins within an electrophoresis gelnetwork according to these techniques is a very slow process and isgenerally unacceptable for preparative purposes.

In response, various additional protocols have been developed which haveattempted to increase the rate of separation, while preserving theaccuracy by which it is performed. There are many types of devicescomprising two or more subcompartments that are separated from eachother by septa, e.g., monofilament screens, membranes, gels, filters,fitted discs, and the like (collectively, “membranes”). Generally, thesedevices are assembled from a plurality of essentially parallel frames orspacers, separated from each other by one or more membranes.

Multi-compartment electrolizerselectrolyzers with isoelectric membraneswere introduced for processing large volumes and amounts of proteins tohomogeneity. For example, see P. G. Righetti, et al., “PreparativeProtein Purification in a Multi-Compartment Electrolyser with ImmobilineMembranes,” 475 J. CHROMATOGRAPHY 293-309 (1989); P. G. Righetti, etal., “Preparative Purification of Human Monoclonal Antibody Isoforms ina Multi-Compartment Electrolyser with Immobiline Membranes,” 500 J.CHROMATOGRAPHY 681-696 (1990); P. G. Righetti, et al., “PreparativeElectrophoresis with and without Immobilized pH Gradients,” 5 ADVANCESIN ELECTROPHORESIS 159-200 (1992). Based on isoelectric focus, thispurification concept progresses under recycling conditions. The proteinmacro-ions are kept in a reservoir and are continuously passed throughan electric field across a multicompartment electrolyzer equipped withzwitterionic membranes.

In this system the protein is always kept in a liquid vein, also calleda “channel.” Consequently, the protein is not lost by adsorption ontosurfaces, as typically occurs in chromatographic procedures. Rather, theprotein is trapped in a chamber that is delimited by two membranes thathave pI values encompassing the pI value of the protein to be separated.Thus, by a continuous titration process, all other impurities, eithernon-isoelectric or having different pI values, are forced to leave thechamber. In the end, the isoelectric/isoionic protein of interest willultimately be present, as the sole species, in the chamber. It should berecognized, however, that the isoelectric and isoionic points of aprotein can differ to some extent in the presence of counterions.

U.S. Pat. No. 4,971,670 describes this process. Isoelectric membranesalso are addressed in U.S. Pat. No. 4,243,507. U.S. Pat. No. 5,834,272describes an immobilization of enzymes that keeps them in solution and,hence, under conditions of homogeneous catalysis. In U.S. Pat. No.4,362,612, adjoining compartments are functionally designed to adjust todifferent pH values electrophoretically, thereby separating dissolvedproteins according to their isoelectric points. Similar multiplesubcompartments devices are described in U.S. Pat. Nos. 4,971,670,5,173,164, 4,963,236, and 5,087,338. Each of these patents discloses adevice, which is comprised of a series of parallel spacers, that areseparated from each other by membranes, that provides an essentiallyparallel array of subcompartments.

Similarly, Amersham Pharmacia markets an IsoPrime filter using aplurality of pI-selective membranes arranged in series. In this devicethe membranes are arranged in ascending or descending pI-selectivity. Asa solution passes through the membranes, molecules having pI valuesbetween two consecutive membranes are trapped between the membranes.However, this process takes on the order of hours to complete.Invitrogen, Inc. markets a device, the ZOOM IEF Fractionator, which issubstantially similar to the IsoPrime device, but which enables themembranes to be individually replaced. However, like the IsoPrime, theZOOM IEF Fractionator process takes on the order of hours to complete.

Various other separation protocols include: sub-cellular fractionation(Lopez, M. F., Electrophoresis, 2000, 21:1082-1093; Hochstrasser, D. F.,et al., Electrophoresis, 2000, 21:1104-1115; Dreger, M., MassSpectrometry Reviews, 2003, 22:27-56; Patton, W. F., J. ChromatographyB, 1999, 722:203-223; McDonald T. G. et al., Basic Res. Cardiol., 2003,98:219-227; Patton, W. F., et al., Electrophoresis, 2001, 22:950-959;Gerner C., et al., Mol. & Cellular Proteomics, 2002, 7:528-537),molecular sizing (Issaq, J. H., et al. 2003, Hochstrasser, et al. 2000),ion exchange (Lopez, M. F., 2000, 17), immobilized metal interactionchromatography (“IMAC”) for calcium binding protein (Lopez, M. F., etal., Electrophoresis, 2000, 21:3427-3440) or phospho-proteins (Hunt, D.F., et al., Nat. Biotechnol., 2002, 20:301-305), hydrophobic (Lopez,2000), heparine (Hochstrasser, et al. 2000) or lectin (Hochstrasser, etal. 2000, Lopez, 2000; Regnier, F., et al., J. Chromatography B, 2001,752:293-306) affinity chromatography, and liquid chromatography (Issaq,J. H., et al 2002, Hochstrasser, et al. 2000).

Two-dimensional liquid chromatography used for intact proteinfractionation, or their trypsic digests, generally uses reverse phase(“RP”) for the second dimension, combined with ion exchange (Yates, J.R., Nature Biotech., 1999, 17:676-682, Unger, K. K., et al., Anal.Chem., 2002, 74:809-820), chromato-focusing (Wall, D., et al., Anal.Chem., 2000, 72:1099-1111), size exclusion (Opiteck, G., Anal. Biochem.,1998, 258:349-361), affinity (Regnier 2001), or another RP (Chicz R., etal., Rapid Commun. in Mass Spectrometry, 2003, 17:909-916) as the firstchromatography step.

Unfortunately, multidimensional chromatography in proteomicfractionation generally never exceeds two dimensions due to high numberof fractions to manage (pH-adjustment, desalting, re-injection in seconddimension) and analyze, especially when a tedious analyticalmethodsmethod such as 2DE makes the final bottleneck. In addition, arelated shortcoming of the prior art is a relative inability to adaptthe various devices to a particular separation protocol. For example, ifa technician desires to identify 20 different proteins within a sample,a system involving only, e.g., eight separation media may beinsufficient. In other words, if a sample contains 20 different proteinsthat have pI values that incrementally vary by 0.1 pH unit, a devicehaving only eight separation media will fail to separate the proteinssufficiently. As a result, the proteins captured by each of theseparation media (e.g., based on pI value) may need to be separated byway of a second separation protocol using the same type of separation(e.g., based on pI value).

What is needed, therefore, is an apparatus and a methodology and anapparatus that address at least one if not more of the deficiencies thatafflict conventional practice, as previously described. Moreparticularly, the need exists for an approach for separating molecules,such as proteins, quickly and accurately.

SUMMARY

An embodiment of the present invention addresses a device that includes,among other possible things, at least three chambers arrayed in a plate.The device has a first face on one side of the plate and a second faceon a second, opposite side of the plate. Each chamber, independent ofany other chamber, has an inlet opening to one face and an outletopening to the other face. A plurality of the chambers are successivelyconnected in series through removable conduits; each conduit connects anoutlet of one chamber with an inlet of another chamber. The series ofchambers and conduits defines a fluid path connecting an inlet of afirst chamber through each of any intermediate chambers to an outlet ofa last chamber.

In a further embodiment of this device, at least some of the conduitsmay pass through the plate and connect outlets opening to the secondface with inlets opening to the first face. Additionally, in a furtherembodiment, all of the conduits may connect outlets opening to thesecond face with inlets opening to the first face.

In another further embodiment of this device, a plurality of thechambers in the series may be arrayed in a linear series (e.g., a row ora column). Each of the chambers may be adjacent a channel that opens toboth faces. The fluid path between at least one outlet and inlet maypass through the channels.

In another further embodiment of this device, at least some of theconduits may connect outlets opening to the first face with inletsopening to the first face, or outlets opening to the second face withinlets opening to the second face. Additionally, in a furtherembodiment, at least one of the conduits may connect an outlet openingto the second face with an inlet opening to the second face. At leastone of the conduits may connect an outlet opening to the first face withan inlet opening to the first face.

In another further embodiment of this device, the conduits may beremovable from the device.

In another further embodiment of this device, at least one chamber inthe series may contain a separation medium. Additionally, in a furtherembodiment, each of the chambers in the series may contain a separationmedium. Moreover, a plurality of the chambers in the series may containdifferent separation media.

In another further embodiment of this device, the series of separationmedia may include, in the direction of the fluid path, either: (a) ahigh selective medium, a medium selective medium, and a low selectivemedium; or (b) a low selective medium, a medium selective medium, and ahigh selective medium.

In another further embodiment of this device, the plurality of chambersin the device may be a multiple of 8.

In another further embodiment of this device, the plurality of chambersin the device may be a multiple of 12.

In another further embodiment of this device, the chambers may bearrayed in at least one liner series.

In another further embodiment of this device, the chambers may bearrayed in a plurality of rows and columns. Additionally, in a furtherembodiment, the chambers may be arrayed in an eight-by-twelve array.

In another further embodiment of this device, the device may furtherinclude a plurality of series of chambers and conduits defining fluidpaths.

In another further embodiment of this device, the device may furtherinclude a collection plate that includes a plurality of wells that arearranged, in rows and columns, to correspond to the chambers of thedevice. Each of the wells of the collection plate may have an inlet.Upon disengagement of the conduits, the inlets of the wells of thecollection plate may be configured to align with the chambers of thedevice.

In another further embodiment of this device, the device may include apump that is configured to push or pull a fluid sample along the fluidpath.

In another further embodiment of this device, the device may furtherinclude a drip-through microtiter plate that includes wellscorresponding to the chambers.

Another embodiment of the invention addresses a device that includes,among other possible things: (a) a plate including, among other possiblethings, at least one row of chambers, each chamber including, amongother possible things, an inlet on a first face of the plate and anoutlet on a second, opposite face of the plate; (b) a removable firstmember that sealingly engages the first face of the plate, wherein thefirst member includes: (I) a plurality of openings aligned with theinlets of a set of odd-numbered wells, and (II) a plurality ofopen-ended conduits aligned with a set of even-numbered wells, whereinthe conduits pass from the inlets to the outlets of the even-numberedwells; (c) a removable second member that sealingly engages the firstmember, wherein the second member includes: (I) an opening aligned withthe inlet of a first chamber, thereby forming the inlet to the firstchamber; and (II) a plurality of conduits, wherein each of the conduitsconnects an opening of the first member to a conduit of the firstmember; (d) a removable gasket that sealingly engages the second face ofthe plate, wherein the gasket includes a plurality of openings alignedwith the outlets of the chambers; and (e) a removable third member thatsealingly engages the gasket, wherein the third member includes aplurality of grooves aligned with the openings in the gasket that,together, form a plurality of conduits, each conduit connecting theoutlet of an odd-numbered well to the outlet of an even-numbered well.The combination of chambers and conduits defines a fluid path thatpasses through odd-numbered wells from inlet-to-outlet and througheven-numbered wells from outlet-to-inlet.

In a further embodiment of this device, each of the odd-numberedchambers may contain a separation medium.

Another embodiment of the invention addresses a device that includes,among other possible things: (a) a plate including, among other possiblethings, at least one row of chambers, each chamber including, amongother possible things, a first opening on a first face of the plate anda second opening on a second, opposite face of the plate, wherein theopenings define inlets and outlets for each of the chambers; (b) aremovable first member that sealingly engages the first face of theplate, wherein the first member includes: (I) an inlet port aligned withthe first opening, which is configured to serve as inlet, of a first ofthe chambers in the row, and (II) a plurality of conduits thatsuccessively connect pairs of the first openings of other chambers inthe row; (c) a removable gasket that sealingly engages the second faceof the plate, wherein the gasket includes a plurality of openingsaligned with the second openings of the chambers; and (d) a removablethird member that sealingly engages the gasket, wherein the third memberincludes a plurality of conduits that successively connect pairs of thesecond openings of other chambers in the row. The combination of wellsand conduits defines a fluid path passing from inlet of the chamber tothe outlet of the last chamber in the row.

In a further embodiment of this device, each of the odd-numbered wellsmay contain a separation medium.

Another embodiment of the invention addresses a device that includes,among other possible things: (a) a plate that includes, among otherpossible things, at least one pair of first and second rows of wells,each well including, among other possible things, an inlet on a firstface of the plate and an outlet on a second, opposite face of the plate;(b) a removable first member that sealingly engages the first face ofthe plate, wherein the first member includes, among other possiblethings: (I) a plurality of openings aligned with the inlets of wells ina first row, thereby defining chambers, and (II) a plurality ofopen-ended channels aligned with wells in a second row, wherein thechannels define conduits passing from the inlets to the outlets of thewells; (c) a removable second member that sealingly engages the firstmember, wherein the second member includes, among other possible things:(I) an opening aligned with an opening in the first member that isaligned with an inlet of a first well in a first row, thereby formingthe inlet to the first chamber; and (II) a plurality of grooves alignedwith the openings of the first member that, together, form a pluralityof conduits, each conduit connecting an n^(th) inlet of a well in afirst row with an n^(th) inlet of a well in a second row; (d) aremovable gasket that sealingly engages the second face of the plate,wherein the gasket includes, among other possible things, a plurality ofopenings aligned with the outlets of the wells; and (e) a removablethird member that sealingly engages the gasket, wherein the third memberincludes, among other possible things: (I) a plurality of groovesaligned with the openings in the gasket that, together, form a pluralityof conduits, each conduit connecting an n^(th) outlet of a well in afirst row with an n+l^(th) outlet of a well in a second row; and (II) anopening aligned with an outlet of a last chamber. The combination ofwells and conduits defines a fluid path passing from inlet to outlet ofthe wells in a first row.

In a further embodiment of this device, the second removable member mayinclude, among other possible things, a first sub-part and a secondsub-part, wherein: (1) the first sub-part includes, among other possiblethings, an opening aligned with the opening in the first member, and (2)the second sub-part includes, among other possible things, an openingaligned with the opening in the first member and a plurality of openingsthat form the grooves when the first sub-part is pressed against thesecond sub-part. In addition, the third removable member may include,among other possible things, a third sub-part and a fourth sub-part,wherein: (1) the third sub-part includes, among other possible things,an opening aligned with the outlet of the last chamber, and (2) thefourth sub-part includes, among other possible things, an openingaligned with the outlet of the last chamber and a plurality of openingsthat form the grooves when the third sub-part is pressed against thefourth subpart.

In another further embodiment of this device, the odd-numbered wells maycontain a separation medium.

Another embodiment of the invention addresses a method that includes,among other possible steps: (a) providing a device that includes, amongother possible things, at least three chambers arrayed in a plate,wherein (i) the device has a first face on one side of the plate and asecond face on a second, opposite side of the plate, (ii) each chamber,independent of any other chamber, has an inlet opening to one face andan outlet opening to the other face; (iii) a plurality of the chambersare successively connected in series through removable conduits, whereineach conduit connects an outlet of one chamber with an inlet of anotherchamber; (iv) the series of chambers and conduits defines a fluid pathconnecting an inlet of a first chamber through each of any intermediatechambers to an outlet of a last chamber; and (v) each chamber in theseries includes a different separation medium; (b) flowing a sampleincluding, among other possible things, a plurality of analytes alongthe fluid path from the inlet of a first chamber through the outlet ofthe last chamber, whereby the separation media capture analytes havingaffinity for the media; (c) removing the conduits that connect outletsto inlets from the device; (d) eluting captured analytes independentlyfrom the chambers; and (e) collecting the eluted analytes.

In a further embodiment of this method, the method may also include thestep of detecting analytes eluted from at least one chamber.Additionally, in a further embodiment of this method, the step ofdetecting analytes may be performed by mass spectrometry or gelelectrophoresis.

In a further embodiment of this method, the step of eluting may includeeluting from at least one chamber in a plurality of fractions.

In a further embodiment of this method, the sample may be selected fromthe group consisting of blood, urine, cerebrospinal fluid andderivatives thereof.

In another further embodiment of this method, the method may furtherinclude the step of (b)(1) separating at least one of the analytescaptured by the separation media into two or more mini-samples.

In another further embodiment of this method, the method may furtherinclude the step of (b)(1) separating at least one of the analytescaptured by the separation media into two or more mini-samples using oneor more of the following protocols: mass spectrometry, isoelectricfocusing, hydrophobicity, and/or hydrophilicity.

Another embodiment of the invention addresses a kit. The kit includes,among other possible things: (a) a device that includes, among otherpossible things: at least three chambers arrayed in a plate, wherein (i)the device has a first face on one side of the plate and a second faceon a second, opposite side of the plate, (ii) each chamber, independentof any other chamber, has an inlet opening to one face and an outletopening to the other face; (iii) a plurality of the chambers aresuccessively connected in series through removable conduits, whereineach conduit connects an outlet of one chamber with an inlet of anotherchamber; and (iv) the series of chambers and conduits defines a fluidpath connecting an inlet of a first chamber through each of anyintermediate chambers to an outlet of a last chamber; and (b) at leastone container containing a separation medium.

Another embodiment of the present invention addresses a method for themultidimensional separation of analytes comprising: (a) separatinganalytes in a sample into a plurality of first aliquots by: (i)providing a first series of different first sorbents arrangedsequentially and in fluid communication from first to last; (ii) flowingthe sample through the first series from first to last so that thesample sequentially contacts the different sorbents, wherein the firstsorbents capture analytes based on a first physical-chemical propertyand the sequence of first sorbents is ordered from first to last indecreasing selectivity with respect to the first physical-chemicalproperty by which the sorbents bind analytes in the sample, whereby eachof the sorbents captures a different set of analytes; and (iii) elutinganalytes from a plurality of the sorbents into the plurality of firstaliquots; (b) separating analytes in each of a plurality of the firstaliquots into a plurality of second aliquots by independently: (i)providing a second series of different second sorbents arrangedsequentially and in fluid communication from first to last; (ii) flowinga first aliquot through the second series from first to last so that thesample sequentially contacts the different sorbents, wherein the secondsorbents capture analytes based on a second, different physical-chemicalproperty and the sequence of second sorbents is ordered from first tolast in decreasing selectivity with respect to the secondphysical-chemical property by which the sorbents bind analytes in thesample, whereby each of the sorbents captures a different set ofanalytes; and (iii) eluting analytes from each of the sorbents into theplurality of second aliquots; and (c) separating the analytes in aplurality of second aliquots by mass spectrometry.

In a further embodiment of this method, the first series of sorbents maybind analytes based on isoelectric point and may be ordered to captureanalytes from highest to lowest isoelectric point or from lowest tohighest isoelectric point.

In another further embodiment of this method, each of the first sorbentsmay include a solid buffer and an ion exchange material. Further, thesecond series of sorbents may bind analytes based on hydrophobic indexand may be ordered to capture analytes from most hydrophobic to leasthydrophobic. Further, each of the second sorbents may include ahydrocarbon chain and an amine ligand. Further, the hydrocarbon chain ofeach sorbent in the series may include more carbons than that of aprevious sorbent.

In another further embodiment of this method, the first series ofsorbents may bind analytes based on hydrophobic index and may be orderedto capture analytes from most hydrophobic to most least hydrophobic.Each of the first sorbents may include a hydrocarbon chain and an amineligand. The hydrocarbon chain of each sorbent in the series may includemore carbons than that of a previous sorbent. The second series ofsorbents may bind analytes based on isoelectric point and may be orderedfrom highest to lowest isoelectric point or from lowest to highestisoelectric point. Each of the second sorbents may include a solidbuffer and an ion exchange material.

In another further embodiment of this method, the mass spectrometry maybe laser desorption/ionization mass spectrometry.

In another further embodiment of this method, the mass spectrometry maybe electrospray mass spectrometry.

Another embodiment of the present invention addresses a device. Thisdevice includes, among other possible things: a plurality ofintersecting row and columns arranged in a plate. Each of the rowsincludes a plurality of sample chambers that are configured to befluidically connected to the other chambers in the row. Each of thecolumns includes a plurality of sample chambers that are configured tobe fluidically connected to the other chambers in the row. At least someof the chambers in one of the rows include chromatographic separationmedia that are configured to capture molecules that have pI valueswithin a given range; the chromatographic separation media of thechambers are sequentially arranged from lowest-to-highest orhighest-to-lowest in the chambers in the row. At least some of thechambers in at least one of the columns include hydrophobic separationmedia that are configured to capture molecules that have hydrophobicityvalues within a given range; the hydrophobic separation media of thechambers are sequentially arranged from lowest-to-highest orhighest-to-lowest in the chambers in the column.

Another embodiment of the present invention addresses a method for themultidimensional separation of analytes. This method includes, amongother possible steps: (a) providing a device comprising a plurality ofintersecting row and columns arranged in a plate, wherein each of therows comprises a plurality of sample chambers that are configured to befluidically connected to the other chambers in the row, wherein each ofthe columns comprises a plurality of sample chambers that are configuredto be fluidically connected to the other chambers in the row, wherein atleast some of the chambers in one of the rows comprise chromatographicseparation media that are configured to capture molecules that have pIvalues within a given range and wherein the chromatographic separationmedia of the chambers are sequentially arranged from lowest-to-highestor highest-to-lowest in the chambers in the row, wherein at least someof the chambers in at least one of the columns comprise hydrophobicseparation media that are configured to capture molecules that havehydrophobicity values within a given range and wherein the hydrophobicseparation media of the chambers are sequentially arranged fromlowest-to-highest or highest-to-lowest in the chambers in the column;(b) providing a sample to a first chamber in the row that comprises thechambers that contain the chromatographic separation media; (c)separating the sample into a plurality of sub-samples respectivelyprovided in each of the chambers of the row by passing the samplethrough the series of chromatographic separation media in the row; and(d) separating the subsample in at least one of the chambers of the rowinto a plurality of mini-samples respectively provided in each of thechambers of the intersecting column by passing the sample through theseries of hydrophobic separation media in the column.

These and other features, aspects, and advantages of the presentinvention will become more apparent from the following description,appended claims, and accompanying exemplary embodiments shown in thedrawings

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a microtiter plate that contains 96chambers arranged in an eight by twelve matrix and that is used in afirst embodiment of the present invention;

FIG. 2A is a cross-sectional view of an upper conduit plate according toa first embodiment of the present invention;

FIG. 2B is a cross-sectional view of the microtiter plate of FIG. 1taken along lines IIB-IIB;

FIG. 2C is a cross-sectional view of a lower conduit plate according toa first embodiment of the present invention;

FIG. 3 is an assembled, cross-sectional view of the upper plate,microtiter plate, and lower plate of FIGS. 2A-2C, respectively;

FIG. 4 is a cross-sectional view of the microtiter plate of FIGS. 1 and3 aligned with a collection plate;

FIG. 5 is a perspective view of a microtiter plate that contains 96chambers arranged in an eight by twelve matrix and that is used in asecond embodiment of the present invention;

FIG. 6, which is a cross-sectional view of the microtiter plate of FIG.5, shows a plurality of conduits that extend from outlets of certainchamber to inlets of other chambers;

FIG. 7, which is a cross-sectional view of microtiter plate of FIG. 6aligned with a collection plate, shows the conduits disconnected fromthe outlets of the chambers;

FIG. 8A is a cross-sectional view of a third embodiment in which anupper plate of the embodiment of FIGS. 1-4 is divided into two separatepieces, thereby enabling the embodiment to act in the manner of theembodiment shown in FIGS. 5-7;

FIG. 8B is an alternate embodiment of the microtiter plate shown in FIG.8A;

FIG. 9A is a top view of a top cover plate according to a fourthembodiment of the present invention;

FIG. 9B is a top view of a top conduit plate according to a fourthembodiment of the present invention;

FIG. 9C is a top view of a top gasket plate according to a fourthembodiment of the present invention;

FIG. 9D is a top view of a microtiter plate, which contains two rows ofchambers and which may be used in a fourth embodiment of the presentinvention;

FIG. 9E is a top view of a bottom gasket plate according to a fourthembodiment of the present invention;

FIG. 9F is a top view of a bottom conduit plate according to a fourthembodiment of the present invention;

FIG. 9G is a top view of a bottom cover plate according to a fourthembodiment of the present invention;

FIG. 9H is a side, cross-sectional view of the plates of FIGS. 9A-9Gtaken along lines indicated by arrows 9H;

FIG. 9I is a side, cross-sectional view of the plates of FIGS. 9A-9Gtaken along lines indicated by arrows 9I;

FIG. 10 is top view of multiple sample pathways arranged side-by-side;

FIG. 11 is a chart of a fractionation of human serum via a series ofchromatographic resins; and

FIGS. 12A-12F show an alternate embodiment multidimensional separationprotocol that is conducted in a matrix of chambers that areinterconnected via a series of valves.

DETAILED DESCRIPTION

Presently preferred embodiments of the invention are illustrated in thedrawings. An effort has been made to use the same reference numbersthroughout the drawings to refer to the same or like parts.

This invention provides a fluidics device comprising a series ofchambers arrayed in a plate and having inlets and outlets connected byremovable conduits. The plate of this invention is, preferably, amicrotiter plate such as drip plate or a filter plate. However, in otherembodiments, the plate may comprise a piece comprising channels, such asbores, that open on either side of the piece and that will definechambers when conduits are attached to the openings of the bores.Preferably, the chambers are arrayed substantially in a plane.

The combination of chambers and conduits define a fluid path whereby afluid can be pumped from chamber to chamber. Preferably, each chamber inthe series comprises a different separation medium that can capture adifferent subset of analytes in a complex sample. A particular utilityof this device is that the conduits are removable so that analytescaptured by a separation medium in any chamber can be isolated by, e.g.,eluting the analytes from the chambers. Once isolated, the analytes canbe detected or analyzed by any available methodology.

Presently preferred embodiments of the invention are illustrated in thedrawings. An effort has been made to use the same, or like, referencenumbers throughout the drawings to refer to the same or like parts.

Each embodiment of the present invention, as hereafter described indetail, may use standard microtiter filtration plates arranged in, e.g.,a typical 96-chamber (eight rows by twelve columns) format. Otherformats also can be used, e.g., plates with any multiple of eightchambers, any multiple of twelve chambers, any multiple of 96 chambers(e.g., a 386 chamber plate), etc. Each chamber of the filtration platemay contain a particular type of separation media, e.g., chromatographicresins (e.g., packed-bed or fluidized-bed).

In a first embodiment (which will later be described with respect toFIGS. 1-4), the fluid is forced alternately down through one chamber inthe series (e.g., a column or a row), up through the next chamber inseries, down through the next chamber in the series, up through the nextchamber in the series, and so on. More specifically, the bottom openingof a first chamber in a series (e.g., column or row) may be connected tothe bottom opening of a second chamber in that series. The top openingof the second chamber may be connected to the top opening of a thirdchamber in that row (or column). The sequencing of connectingbottom-to-bottom and top-to-top generates a flow path that travels fromtop-to-bottom of the first chamber, then bottom-to-top of secondchamber, and then top-to-bottom of third chamber, etc.

In a second embodiment (which will later be described with respect toFIGS. 5-7), the fluid is forced down through each chamber in the series.More specifically, the bottom opening of a first chamber in a column (orrow) may be connected to the top opening of a second chamber in thatcolumn (or row). The bottom opening of the second chamber may beconnected to the top opening of a third chamber in that row (or column).The sequencing of connecting bottom-to-top generates a flow path thattravels from top-to-bottom of the first chamber, top-to-bottom of secondchamber, top-to-bottom of third chamber, etc.

In other embodiments, these arrangements can be mixed, including bothbottom-to-bottom/top-to-top and bottom-to-top connections.

With any of the aforementioned embodiments, a series of parallelseparation protocols may simultaneously occur along each of the columns(or rows). In other words, different samples can be provided to thefirst chamber in given column (or row); each of the samples may thenpass through each of the chambers (and the varying separation mediatherein) in its respective row. Each of the separation media throughwhich a particular sample passes serves to capture a portion of thesample such that upon reaching the final chamber in the given row,various portions of the sample will be retained in each of the chambersthrough which the sample passed. Moreover, upon completion of thesample's separation, each of the sample portions (or “sub-samples”) maybe transferred to a collection plate for, if desired, further analysis(e.g., mass spectrometry, gel electrophoresis, etc.) or separation.

More specifically, each sample may be introduced to the device via thefirst chamber of a particular column (or row). The sample may then bepumped through the device via, e.g., a peristaltic pump (at a variety ofsuitable flow rates), until the sample passes through all separationmedia in each of the chambers in that column (or row). After the sampleis bound across each of the separation media, all connecting tubesand/or conduits may be removed. As a result, the device may serve as atypical sample-filled filter plate operating in batch or continuous flowmode. However, each of the separation media may be, if desired,processed independently of any other separation media. Moreover, thesample can be removed from each of the chambers of the microtiter plateby, e.g., vacuum or centrifugation.

The first embodiment will now be described with respect to FIGS. 1-4. Asshown in FIG. 1, a microtiter plate 100 includes a plurality of chambers102 that are arranged in a matrix of rows and columns. Each of thechambers 102 is substantially in a plane that is defined by upper andlower faces 110, 112 of the microtiter plate 100. As shown, themicrotiter plate 100 may include, e.g., 96 chambers arranged in an eightby twelve matrix. Neither the number of chambers 102 nor the number ofrows/columns, however, is critical to the invention. Rather, anysuitable number of chambers 102 can be used. Moreover, a technician maychoose a microtiter plate based on the number of chambers therein and acorresponding number of chambers needed for a particular separationprotocol.

FIG. 2B shows a cross-section of the microtiter plate 100. As shown inFIG. 2B, each of the chambers 102 of the microtiter plate 100 has twoopenings 104, one of which serves as an inlet 106 and the other of whichserves as an outlet 108. Whether an opening 104 of a particular chamber102 serves as inlet 106 or as an outlet 108, however, will depend on thedirection of flow through the chamber 102, as later described in detail.That is to say, whether a particular opening in a chamber is an inlet oran outlet is independent of the face on which the opening opens.

Positioned above and below the microtiter plate 100, are first andsecond conduit plates 120, 140, respectively. The first conduit plate120 (shown in FIG. 2A) includes an inlet port 122 and an outlet port126. Between the inlet and outlet ports 122, 126, is a series ofconduits 124. As shown, for example, the first plate 120 may have threeconduits 124A, 124B, 124C that correspond to a particular set (e.g., arow) of chambers 102 in the microtiter plate 100.

Similar to the first plate 120, the second plate 140 also contains aplurality of conduits 144. As shown, the second plate 140 may contain,e.g., four conduits 144A, 144B, 144C, 144D that correspond to aparticular set (e.g., a row) of chambers 102 in the microtiter plate100. In addition, although the shown embodiment of the second plate 140does not include inlet and/or outlet ports, other embodiments of theinvention (e.g., where a separation will involve an odd number ofchambers) may include an outlet (or inlet) in the second plate 140.

Some versions of the embodiment may also include a gasket 160 that isconfigured to be positioned between the lower plate 140 and themicrotiter plate 100. The gasket 160 may serve to seal the lower plate140 to the microtiter plate 100.

When the first and second plates 120, 140 are connected (and possiblysealed by the gasket 160) to the upper and lower faces 110, 112,respectively of the microtiter plate 100, a fluid pathway (which isindicated by arrows in FIG. 3) is established. The fluid pathway extendsbetween, e.g., a first chamber 102A in a row and a last chamber 102H inthat row. More specifically, a sample can pass through the inlet port122 in the first plate 120 and through the inlet 106A of a first chamber102A. A sub-sample of the sample may be retained within the firstchamber 102A by, e.g., a separation media (not shown) provided therein.The sample remainder, passes through the outlet 108A of the firstchamber 102A, though a first conduit 144A in the second plate 140, andthrough the input 106B of a second chamber 102B. Subsequently, anothersub-sample may be retained within the second chamber 102B by means of adifferent separation media (not shown) provided in the second chamber102B. Similarly, that which remains of the original sample (afterpassing through the separation media in the first and second chambers102A, 102B) passes through the output 108B of the second chamber 102B,through a conduit 124A in the first plate 120, and through the inlet106C of a third chamber 102C in which another sub-sample may beretained.

This iterative process continues until that which remains of the samplepasses through a separation media (not shown) in the last chamber 102H.At this point, depending on the embodiment, the remaining sample may beremoved from the last chamber 102H by means of the outlet port 126 inthe first plate 120. In other embodiments, however, the separation maycontinue into a different row (or column) by either: (a) providinganother conduit 124 (not shown) in the first plate 120 that connects theoutlet 108H of the last chamber 102H with the inlet 106 of a chamber 102in the different row (or column); or (b) connecting the outlet port 126in the first plate 120 to an inlet 106 of a chamber 102 in the differentrow (or column).

Regardless of the number of chambers 102 through which a sample passes,numerous means exist to aid the movement of the sample. For example, apump (e.g., a peristaltic pump) could be connected to the inlet port 122of the first plate 120; this pump could push the sample through thechambers 102. Alternatively (or additionally), a pump (e.g., aperistaltic pump) could be connected to the outlet port 126 of the firstplate 120; this pump could pull the sample through the chambers 102.

Other related devices of this embodiment contemplate compound (or“bidimensional”) separation protocols. For example, if the first andsecond plates 120, 140 were used to separate a sample into eightsub-samples (e.g., in chambers 102A-102H) in a particular row, each ofthose sub-samples could be further separated along its respective columninto a plurality (e.g., twelve) of mini-samples.

In one such embodiment, the first and second plates 120, 140 could bereplaced with alternate plates (not shown) that contain conduits thatconnect chambers within a particular column of the plate. As a result,if the first separation protocol (using the first and second plates 120,140) were along a particular row and were based on a first separationprotocol, each of the remaining chambers in the various columns could beused for further separation based on a second separation protocol. Inother words, each of the sub-samples could flow through separation mediain the chambers of their respective columns. As a result, if, e.g., aneight by twelve matrix of chambers were employed, after a firstseparation protocol, eight sub-samples could be obtained. And, after asecond separation protocol, 96 individual mini-samples could beobtained.

In addition to the foregoing compound separation, each of themini-samples in, e.g., the chambers in a particular column could betransferred (by means later described in detail) to a like-sized columnof another microtiter plate (not shown) where they could be furtherseparated, by means of a third separation protocol, along the rowsthereof. Alternatively or additionally, each of the individualmini-samples could be further analyzed and/or separated using anyconventional other analytical or separation protocol (e.g.,chromatography, isoelectric focusing, hydrophobicity, hydrophilicity,size exclusion, mass spectrometry, gel electrophoresis, ion exchange,various other separation protocols (some of which are later described asembodiments of the present invention), etc.), thereby adding anotherdimension to the separation of the original sample. Moreover, if, e.g.,an additional chromatography separation protocol were to be employed,any conventional chromatography technique (e.g., ion exchanges,hydrophobic solid phases, affinity sorbents, metal chelating resins, gelfiltration material, hydroxyapatite crystals, etc.) may be used.

To move each of the mini-samples of, e.g., a row, a column, or a matrixto a more permanent storage device, a collection plate 200 of the typeshown in FIG. 4 could be employed. In this embodiment, the second plate140 (with or without the gasket 160) may be detached from the microtiterplate 100 and the openings 104 on the lower side of the microtiter plate100 may be aligned with wells 202 in the collection plate 200. Thesub-samples (or mini-samples after a compound separation) may pass intothe wells 202 of the collection plate 200 by any conventional means,e.g., force of gravity, vacuum, etc.

Similarly, in other related embodiments, the top plate 120 may beremoved to enable one or more solvents to be added tosub-samples/mini-samples (e.g., analytes) housed within the chambers 102via the chambers' upper openings 104. Moreover, the solvent(s) could beused to facilitate draining the sub-samples/mini-samples through thechambers' lower openings 104 of the chambers 102 (i.e., thesub-samples/mini-samples could drip out of the chambers 102 and into,e.g., a collection plate 200).

Another embodiment of the present invention will hereafter be discussedwith respect to FIGS. 5-7. In this embodiment, separable conduits 502connect outlets-to-inlets of various chambers 510 in, e.g., a standardeight by twelve microtiter plate 500. As shown in FIG. 5, one advantageof this embodiment is that a technician can readily connect as manychambers 510 as desired. More specifically, as shown, a technician couldconnect an inlet port 512 to an inlet 506 of a first chamber 510A.Subsequent conduits 502 may then connect, e.g. a total of sixteenchambers 510-A to 510-P in series. Finally, an outlet 508 of the lastchamber 510-P in the series may be connected to an outlet port 514.Adjacent each of the chambers 510-A to 510-P in the linear series inwhich a separation is to occur, there is provided an access channel520-A to 520-P, which may, in some embodiments, be another chamber thatis not being used to conduct a separation protocol.

As shown in FIG. 6, the access channels 520-A to 520-P enable conduits502 to pass through the microtiter plate 500, thereby serving to connectthe outlet 508 of one chamber (e.g., 510A) on one (lower) side of theplate 500 with the inlet 506 of a second chamber (e.g., 510B) on asecond (upper) side of the plate 500. As a result of this flexiblestructure, a technician may readily connect as many chambers 510 asdesired. Moreover, when the chambers 510 are connected, a sample mayflow top-to-bottom therethrough in manner that is generally in the shapeof a square-wave.

Regardless of the number of chambers 510 through which a sample passes,numerous means exist to aid the movement of the sample. For example, apump (e.g., a peristaltic pump) could be connected to the inlet port 512of the first chamber 510A; this pump could push the sample through thechambers 510-A to 510-P. Alternatively (or additionally), a pump (e.g.,a peristaltic pump) could be connected to the outlet port 514 of thelast chamber 510-P; this pump could pull the sample through the chambers510-A to 510-P.

Each of the sub-samples (or mini-samples for compound separations) maybe transferred to a chamber in another microtiter plate where they maybe more permanently stored, further analyzed (e.g., using massspectrometry, gel electrophoresis, etc.), and/or separated (e.g., usinghydrophobicity). To move each of the sub-samples (or mini-samples) to amore permanent storage device, a collection plate 200 of the type shownin FIG. 7 may be employed. In this embodiment, the conduits 502 may bedetached from the outlets 508, which, in turn, may be aligned with wells202 in the collection plate 200. The sub-samples (or mini-samples aftera compound separation) may pass into the wells 202 of the collectionplate 200 by any conventional means, e.g., force of gravity, vacuum,etc.

Similarly, in other related embodiments, the conduits 502 may be removedto enable one or more solvents to be added to sub-samples/mini-samples(e.g., analytes) housed within the chambers 510 via the chambers' inlets506. Moreover, the solvent(s) could be used to facilitate draining thesub-samples/mini-samples through the chambers' outlets 508 (i.e., thesub-samples/mini-samples could drip out of the chambers 510 and into,e.g., a collection plate 200).

Similar to the aforementioned embodiment of FIGS. 1-4, when a technicianhas separated a sample into a plurality of sub-samples through a firstseparation protocol using the microtiter plate 500, the technician mayperform a second separation protocol. Through the second separationprotocol, one or more of the sub-samples may be further separated into aplurality of mini-samples.

FIG. 8A is a cross-sectional view of an alternate embodiment in whichthe upper plate 120 of the embodiment to FIGS. 1-4 is divided into twoseparate pieces 120A, 120B, thereby creating a device that has a similartop-to-bottom functionally as the embodiment shown in FIGS. 5-7. Morespecifically, long capillary portions 128 of the lower piece 120B areconfigured to pass entirely through a chamber 102, i.e., each longportion 128 essentially enables a sample to bypass the chamber 102 inwhich the long portions 128 is provided. In other words, as a result ofthe long capillary portions 128, a sample may pass from an outlet 108 ofa first chamber, through a conduit 144 in the lower plate 140 anddirectly through a second chamber 102 in which the long portion 128 ispositioned. As a result, the sample can directly pass to the inlet 106of a third chamber 102 (via a conduit 124 in the upper plate 120A).Accordingly, this embodiment is a top-to-bottom device similar to thatshown in FIGS. 5-7.

In a further version of the embodiment shown in FIG. 8A, one or moregaskets 160 may be provided (as shown) to enhance the seal between thevarious plates 120A, 120B, 100, 140.

FIG. 8B shows an alternate embodiment plate 500′. Like the embodimentshown in FIG. 8A, in this embodiment, the conduits 502 of the embodimentshown in FIGS. 5-7 are replaced by a series of plates 550, 560, 580,which are shown as being separated for ease of understanding. In use,the plates 500, 550, 560, 580 would be sandwiched together to create atop-to-bottom fluid pathway that, like the embodiment shown in FIG. 8A,is generally in the shape of a square-wave.

In this alternate embodiment, an upper plate 550 contains an inlet port552, a plurality of conduits 554, and an outlet port 556. The inlet port552 is configured to align with the an inlet 562 of an inlet channel 566of a channel plate 560 and, in turn, with an inlet 506A of a firstchamber 510A in a linear series. Similarly, the outlet port 556 isconfigured to align with an outlet 508′ of an outlet channel 564 of thechannel plate 560 and, in turn (via a conduit 584F below described) withan outlet 508F of a last chamber 510F in the linear series. The conduits554 in the upper plate 550 are configured to connect outlets 508′ ofoutlet channels 564 to inlets 506′ of inlet channels 566 in the channelplate 560. A lower plate 580, which contains a plurality of conduits584, is positioned on the other side of the microtiter plate 500′. As aresult, a sample can pass: (a) from the outlet 508A of, e.g., the firstchamber 510A, (b) though a conduit 584A in the lower plate 580 to anoutlet channel 564A in the channel plate 560, (c) through the outletchannel 564A, (d) through a conduit 554A in the upper plate 550, and (e)into the inlet 506B of a second chamber 510B. As a result, the sampletakes a top-to-bottom (generally square-wave shaped) path through eachof the chambers 510.

In contrast to the long capillary portions 128 of the embodiment shownin FIG. 8A, in this embodiment, chamber portions 568 of the channelplate 560 are sized to substantially fill chambers, which serve aschannels, in the microtiter plate 500′. As a result, each of the outletchannels 564 in the channel plate 560 may be have a diameter that isdifferent than the diameter of the long capillary portions 128 of theembodiment shown in FIG. 8A. Moreover, as a result of the chamberportions 568, the system may enjoy better overall sealing conditions.

In should be readily recognized that this embodiment microtiter plate500′ can also employ a gasket 570 (as shown). Moreover, the top andbottom plates 550, 580 could be formed in multiple parts, which wouldfacilitate cleaning of the conduits 554, 584 therein.

Similar to the aforementioned embodiments, when a technician hasseparated a sample into a plurality of sub-samples through a firstseparation protocol using the microtiter plates of the embodiments shownin FIG. 8A or 8A, the technician may perform a second separationprotocol. Through the second separation protocol, one or more of thesub-samples may be further separated into a plurality of mini-samples.

FIGS. 9A-9I depict another embodiment of the present invention. In thisembodiment, a plate 1100 (FIG. 9D), which contains a plurality ofchambers 1102, may be combined with an upper cover plate 610 (FIG. 9A),an upper conduit plate 600 (FIG. 9B), a lower conduit plate 700 (FIG.9F), and a lower cover plate 710 (FIG. 9G) to create a flow path througha plurality of chambers 1102A-1102I that are positioned in two adjacentrows. In addition, a channel plate 730 (FIG. 9C) may be provided betweenthe plate 1100 and the upper conduit plate 600. Similarly, a gasket 740(FIG. 9E) may be provided between the plate 1100 and the lower conduitplate 700.

In this embodiment, an inlet port 612 in the upper cover plate 610 maybe aligned with an inlet port 602 in the upper conduit plate 600 and aninlet port 732 in the channel plate 730. Together, the inlet ports 602,612, 732 may form an inlet to a first chamber 1102A in a first row R1. Asample supplied to the first chamber 1102A (via the inlet ports 602,612, 732) may pass top-to-bottom through the first chamber 1102A andinto a first diagonal conduit 708A in the lower conduit plate 700. Fromthe first diagonal conduit 708A, the sample (or portion thereof) mayflow into a channel 734A in a second chamber 1102B (which may or may nothave a separation medium therein) that is in a second row R2, which isbehind the first row R1. After passing bottom-to-top through the channel734A in the second chamber 1102B, the sample (or portion thereof) mayflow through a first transverse conduit 606A (in upper conduit plate600) to a third chamber 1102C that, like the first chamber 1102A, is inthe first row R1. The sample may then pass top-to-bottom through thethird chamber 1102C and continue on this front-to-back/bottom-to-top,back-to-front/top-to-bottom path until it reaches a final chamber 11021,which (as shown) may be in the first row. The sample (or portionthereof) may then be removed from the final chamber 11021 by way ofoutlet ports 702, 712 formed in the lower conduit plate 700 and thelower cover plate 710, respectively. Together the outlet ports 702, 712may serve as an outlet.

In some embodiments, the upper cover plate 610 and the upper conduitplate 600 may be integrally formed. Additionally or alternatively, thelower cover plate 710 and the lower conduit plate 700 may be integrallyformed.

Another related embodiment of the invention is shown in FIG. 10. In thiscase, each chamber in a first linear series of chambers (e.g., a columnor a row) in a plate 1200 contains a separation medium through which asample flows in the same direction (e.g., top-to-bottom), and thechambers in an adjacent linear series serve as channels though which thesample flows in the opposite direction (e.g., bottom-to-top). In thisembodiment, for example, a conduit, such as a tube, may be connected theoutlet at the bottom of a first chamber 1A in a first linear series, maypass up through a second chamber 2B in a second linear series, and mayconnect to the inlet at the top of third chamber 1B in the first linearseries. Another conduit may connect the outlet at the bottom of thethird chamber 1B, pass up through a fourth chamber 2C in the secondlinear series, and may connect to the inlet at the top of a fifthchamber 1C in the first linear series, and so forth. The diagonalarrangement of the conduits can be located at the top of the plate(similar to the embodiment shown in FIGS. 9A-9I), if desired.

Although FIG. 10 shows multiple separation protocols 10A-10F on the sameplate 1200, this parallel functionality is not exclusive to thisembodiment. Rather, any of the previously described embodiments may,like that of the embodiment shown in FIG. 10, involve parallelseparation protocols on one plate.

In some alternate embodiments of any of the aforementioned embodiments,the flow directions across the various separation media may be operatedin packed-bed format, fluidized-bed format, or a combination ofpacked-bed and fluidized-bed, depending on the configuration of theconnection conduits.

It should be readily recognized that the separation media are notlimited to any particular type. Rather, the separation media can be ofany type of including, but not limited to solid materials (both porousand non-porous) such as beads, irregular particles, membranes,monoliths, etc.

By way of example, a sample solution, which contains a complex mixturethat includes a plurality of different biomolecular components, may beintroduced into a first chamber 102/510 in a series of chambers for atleast partial resolution as described hereinbelow. Each of the chambers102/510 through which the sample passes may, e.g., include a differentsorbent material. More specifically, the sorbent materials may be chosensuch that substantially all of the distinct biomolecular components ofthe sample are captured by the various sorbents in the chambers 102/510.After the capture of the various subsets of the plurality ofbiomolecular components, the sorbents, including the capturedbiomolecular components, may be isolated (i.e., removed from thechambers) or otherwise removed for further processing, as previouslydiscussed.

As used herein “capture” refers to the capacity of a sorbent to attractand reversibly retain one or more biomolecular components in a samplesolution such that certain subsets-samples of the biomolecularcomponents are substantially completely removed from the sample solutionduring passage through the chambers 102/510. Those of skill in the artof separating mixtures of chemicals of biological origin, such asprotein purification, will appreciate that a sorbent's capacity toretain a biomolecular component inherently includes a specificity of thesorbent for certain biomolecular components that is defined by theinteraction between the sorbent and a biomolecular component under theambient conditions in which the sorbent and the solution are in contact(e.g., the temperature and ionic strength or pH of the solution beingpassed through the chambers 102/510). The interaction can be anyphysicochemical interaction known or believed to be sufficient to causesorption of a biomolecular component (or subset-sample of biomolecularcomponents) by the sorbent to substantially completely deplete thesolution of the biomolecular component (or subset-sample), but stillallow subsequent elution of the captured biomolecular component(s).

Typical sorbent-biomolecular component interactions include withoutlimitation: ion exchange (cation or anion); hydrophobic interactions;biological affinity (including interactions between dyes and ligandswith proteins, or lectins with glycoconjugates, glycans, glycopeptides,polysaccharides, and other cell components); immunoaffinity (i.e.,antigen-antibody interactions or interactions between fragmentsthereof); metal-chelate or metal-ion interactions, interactions betweenproteins and thiophilic materials, interactions between proteins andhydroxyapatite, and size exclusion. Many such materials are known tothose having skill in the art of protein or nucleic acid purification.These materials can be made using known techniques and materials orpurchased commercially. Descriptions of these materials and examples ofmethods for making them are described in “Protein PurificationProtocols” 2d Edition, Cutler, Ed. Humana Press 2004, which isincorporated herein by reference in its entirety for all purposes.

Ion exchanging materials include strong and weak cation- and anionexchange resins. Strong, some of which are later listed in Table 4.Examples of strong cation exchanging ligands include sulfopropyl (SP)and methyl sulfonate (S). WeakExamples of weak cation exchange ligandsinclude carboxymethyl (CM). StrongExamples of strong anion exchangeligands include quaternary ammonium and quaternary aminoethyl (QAE).WeakExamples of weak anion exchange ligands include diethylaminoethyl(DEAE). Examples of suitable ion-exchange materials include withoutlimitation, the materials sold commercially under the trade names: Q-,S-, DEAE- and CM CERAMIC HYPERD®; DEAE-, CM-, and SP TRISACRYL®; M-,LS-; DEAE-, and SP SPHERODEX® LS; and QMA SPHEROSIL® LS from PallCorporation (East Hills, N.Y. (Fremont, Calif.). Other suitable ionexchanging materials are sold under the trade names: UNOSPHERE,MACRO-PREP (including HIGH Q, HIGH S, DEAE, and CM), and AG and Bio-Rexfrom Bio-Rad Laboratories of Hercules, Calif. Still more suitablecommercially available ion exchange materials are sold under the tradenames: DEAE-TRISACRYL®, DEAE SEPHAROSE®, DEAE-CELLULOSE,DIETHYLAMINOETHYL SEPHACEL®, DEAE SEPHADEX®, QAE SEPHADEX®, AMBERJET®,AMBERLITE®, CHOLESTYRAMINE RESIN, CM SEPHAROSE®, SP SEPHAROSE®,SP-TRISACRYL®, CELLULOSE PHOSPHATE, CM-CELLULOSE, CM SEPHADEX®, SPSEPHADEX®, and AMBERLITE® from Sigma-Aldrich Co. (St. Louis, Mo.). Othercommercial sources for ion exchange materials include AmershamBiosciences (www.amersham.com). Still other materials will be familiarto those having skill in the art of protein purification.

Materials suitable for exploiting hydrophobic interactions (hydrophobicinteraction chromatography, “HIC”) include those sold under the tradenames: PHENYL SEPHAROSE 6 FAST FLOW, BUTYL SEPHAROSE 4 FAST FLOW, OCTYLSEPHAROSE 4 FAST FLOW, PHENYL SEPHAROSE HIGH PERFORMANCE, PHENYLSEPHAROSE CL-4B, OCTYL SEPHAROSE CL-4B, SOURCE™ 15ETH, SOURCE 15ISO, andSOURCEPHE from Amersham Biosciences of Piscataway, N.J. Also availableare materials sold as FRACTOGEL® EMD PROPYL (S) AND FRACTOGEL® EMDPHENYL I (S) from VWR International (www.chromatography.uk.co). Stillother commercially available HIC materials include the materials soldunder the trade names: TOYOPEARL and TSKGEL from Tosoh Bioscience LLC ofMontgomeryville, Pa. An equivalent material is sold commercially underthe trade name MEP HYPERCEL by Pall Corporation (East Hills, N.Y.).Still other materials will be familiar to those having skill in the artof protein purification.

Affinity materials include any materials effective to attract and sorbbiomolecular components on the basis of structural interactions betweena biomolecular component and a ligand such as: antibody-antigen,enzyme-ligand, nucleic acid-binding protein, and hormone-receptor. Theinteractions can be between naturally occurring or synthetic ligand anda biomolecular component. The ligands can be either mono-specific (e.g.,a hormone or a substrate) or group-specific (e.g., enzyme cofactors,plant lectins, and Protein A). Examples of common group-specific ligandssuitable for the present invention are provided in Table 1.

TABLE 1 Ligand(s) Target(s) 5′-AMP, 5′-ATP Dehydrogenases NAD, NADPDehydrogenases Protein A Immunoglobulins Protein G ImmunoglobulinsLectins Polysaccharides, Glycoproteins Histones DNA HeparinLipoproteins, DNA, RNA, clotting factors Gelatin Fibronectin attachmentfactors Lysine rRNA, dsDNA, Plasminogen Arginine Fibronectin attachmentfactors Benzamidine Serine proteases Polymyxin Endotoxins CalmodulinKinases Cibacron Blue Kinases, Phosphatases, Dehydrogenases, AlbuminsBoronic Acid Biomolecules containing cis- diols (RNA, glycoproteins)

Thus, a wide variety of biomolecular materials can be sorbed usingaffinity materials. Commercially available affinity materials includethose sold under the trade names: PROTEIN A CERAMIC HYPERD® F, BLUETRISACRYL® M, HEPARIN HYPERD® M, and LYSINE HYPERD® from PallCorporation (East Hills, N.Y.). Still other commercially availablematerials are provided by commercial suppliers including AmershamBiosciences (www.amershambiosciences.com) and Sigma-Aldrich(www.sigmaaldrich.com). Still other materials will be familiar to thosehaving skill in the art of protein purification.

In some embodiments of the invention, the affinity materials are derivedfrom reactive dyes are used to create sorbents. Dye-ligand sorbents areoften useful for binding proteins and enzymes that use nucleic acidcofactors, such as kinases and dehydrogenases. But, other proteins,including serum albumins, can be sorted efficiently with these sorbentsas well. Examples of suitable commercially available materials includethose sold under the trade names REACTIVE BLUE, REACTIVE RED, REACTIVEYELLOW, REACTIVE GREEN, and REACTIVE BROWN (Sigma-Aldrich); DYEMATRIXGEL BLUE, DYEMATRIX GEL RED, DYEMATRIX GEL ORANGE, and DYEMATRIX GELGREEN (Millipore, Billerica, Mass.); and the Procion dyes known as BlueH-B (Cibacron Blue), Blue MX-R, Red HE-3B, Yellow H-A, Yellow MX-3r,Green H-4G, Green H-E4BD, Brown MX-5BR. Still others will be familiar tothose having skill in the art of protein purification.

Useful sorbents can also be constructed from lectins to separate andisolate glycoconjugates, glycans, glycopeptides, polysaccharides,soluble cell components, and cells. Suitable lectins include those shownin Table 2.

TABLE 2 Lectin Use(s) Concanavalin A Separation of glycoproteins,glycoprotein enzymes, and lipoproteins; isolation of IgM Lens culinarisIsolation of gonadotropins, mouse H antigens, detergent-solubilizedglycoproteins Tritium vulgaris Purification of RNA polymerasetranscription cofactors Ricins communis Fractionation of glycopeptide-binding proteins Jacalin Purification of C1 inhibitors, separation ofIgA1 and IgA2 Bandeira simplicifolia Resolution of mixtures ofnucleotide sugars

Commercially available lectins include those sold under the trade names:AFFI 10, AFFI 15, AFFI PREP 10, and AFFI PREP 15 (Bio-Rad, Hercules,Calif.); CNBR ARGINOSE, EPDXY-ACTIVATED ARAGROSE, CDI AGAROSE, andPOLYACRYLHYDRAZIDE AGAROSE (Sigma-Aldrich, St. Louis, Mo.); and REACTI6X (Pierce, Rockford, Ill.).

Immunoaffinity materials can be made using standard methods andmaterials known to those having skill in the protein purification arts(see, e.g., “Protein Purification Protocols”). Commercially availableimmunoaffinity material include those sold by Sigma-Aldrich(www.sigmaaldrich.com) and Amersham Biosciences (www.amersham.com).Similarly, metal-ion affinity (IMAC) materials can be prepared usingknow materials and methods (see, e.g., “Protein PurificationProtocols”), or purchased commercially (e.g., from Sigma-Aldrich(www.sigmaaldrich.com) or Amersham Biosciences (www.amersham.com)).Common metal include Ni(II), Zn(II), and Cu(II). Some examples of thesematerials are shown Table 3.

TABLE 3 Chelator Ligand Metal Iminodiacetate (IDA) Transition Metals2-Hydroxy-3-[N-(2- Transition Metals pyridylmethyl)glycine]propylα-Alkyl nitrilotriacetic acid Transition Metals Carboxymethylatedaspartic acid Ca⁺² Ethylenediamine (TED) Transition Metals (GHHPH)_(n)G*Transition Metals *The letters G and H refer to standard amino acidnotation: G = glycine, and H = histidine.

The synthesis of hydroxyapatite (HT/HTP) and thiophilic (TAC) sorbentswill also be familiar those having skill in the protein purificationarts (see, e.g., “Protein Purification Protocols”). Commercial sourcesinclude Bio-Rad of Hercules, Calif. (trade name CHT), Pall Corporation(East Hills, N.Y.) (trade name HA ULTROGEL®), and Berkeley AdvancedBiomaterials of San Leandro, Calif. (trade name HAP). Thiophilicsorbents also can be made using methods and materials known in the artor protein purification or purchased commercially under the trade names:MEP HYPERCEL (Ciphergen Biosystems, Fremont, Calif.), THIOPHILIC UNIFLOWand THIOPHILIC SUPERFLOW (Clonetech, Palo Alto, Calif.), THIOSORB(Millipore, Billerica, Mass.), T-GEL (Affiland, Ans-Liege, Belgium),AFFI-T (Ken-en-Tec, Copenhagen, Denmark), HI-TRAP (Amersham Biosciences,Piscataway, N.J.), and FRACTOGEL (Merck KgA, Poole Dorset UK).

The above-described sorbent materials have different degrees ofspecificity for different biomolecular components. In this regard, theterm “specificity” relates to the number of different biomolecularspecies in a given sample that a sorbent can bind. In one aspect,sorbents can be grouped by their relative degrees of specificity, e.g.,high specificity sorbents, moderate specificity sorbents, and lowspecificity sorbents.

High specificity materials include those materials that generally have astrong preference to sorb certain biomolecules or subsets ofbiomolecules. Often such materials include highly biospecific sorptioninteractions, such as antibody-epitope recognition, receptor-ligand, orenzyme-receptor interactions. Examples of these sorbents include ProteinA-, Protein G-, antibody-, receptor- and aptamer-bound sorbents.Moderate specificity sorbents include materials that also have a degreeof biospecific interactions but to a lesser degree than high specificitysorbents, and include: MEP, MBI, hydrophobic sorbents, and heparin-,dye-, and metal chelator-bound materials. Many “mixed-mode” materialshave moderate specificity. Some of these bind molecules through, forexample, hydrophobic and ionic interactions. Low specificity materialssorbents include materials that sorb bimolecular components using bulkmolecular properties (such as acid-base, dipole moment, molecular size,or surface electrostatic potential) and include: zirconia, silica,phenylpropylamine cellulose, ceramics, titania, alumina, and ionexchangers (cation or anion).

In one embodiment of the invention, the solution of biomolecularcomponents may be contacted with at least two different sorbents fromamong high-, moderate-, or low-specificity sorbents. In someembodiments, the solution will be contacted with one, two, or three ormore materials of the same degree of specificity (e.g., two materials ofmoderate specificity or three materials of low specificity). In anotherembodiment, the solution may be contacted with a plurality of sorbentsthat define a progression from high specificity to low specificity. Inanother embodiment, the solution may be contacted with a plurality ofsorbents that define a progression from high specificity to lowspecificity. In yet another embodiment, the sorbent materials may bearranged to provide a substantially linear progression of specificities.In still another embodiment, the sorbent materials may form asubstantially contiguous body. In still another embodiment, the sorbentsmay be mutually orthogonal, i.e., the capacity of each sorbent may besubstantially selective for a unique biomolecular component or subset ofbiomolecular components. In another embodiment, the sorbents may bechosen such that at least one sorbent is a high specificity sorbent andat least one other sorbent is either a moderate- or low specificitysorbent. In yet another embodiment, the sorbents may be chosen such thatat least one sorbent each is a high specificity sorbent, a moderatespecificity sorbent, and low specificity sorbent. In still anotherembodiment, at least two sorbents may be chosen from two classes of highspecificity sorbents, moderate specificity sorbents, and low specificitysorbents. In another embodiment, at least two sorbents may be highspecificity sorbents and at least one sorbent may be a low specificitysorbent.

The progression from high specificity to low specificity serves aparticularly useful purpose. In particular, it allows fractionation ofthe proteins in the sample into largely exclusive groups, but ofdecreased complexity. As such, the proteins in the various fractions maybe more easily resolved. For example, a low- or moderate-specificityresin might have affinity for or bind to many biomolecules in a sample,including ones in very high concentration. However, by exposing thesample to a high specificity sorbent that is directed to the protein inhigh concentration before exposing to the moderate-specificity sorbent,one can remove most or all of the high concentration protein. In thisway, the set of biomolecules captured by the moderate specificitysorbent will largely or entirely exclude the high concentrationbiomolecule. This results in a less complex set of proteins captured bythe moderate specificity sorbent. The strategy, thus, is to remove atearlier stages biomolecules, e.g., proteins, that would otherwise becaptured by sorbents at later stages of the fractionation process sothat at each chamber 102/510, the complexity of the biomolecules passingto the next chamber 102/510 is decreased.

In one embodiment, the sorbents may be chosen such that the biomolecularmaterials of the greatest concentrations are removed first. For example,arranging the sorbents such that a Protein A sorbent and a Cibacron Bluesorbent are the first two sorbents can reduce the dynamic range of humanserum. Ninety percent of the protein composition of human serumincludes: albumin, IgG, transferrins, α-1 anti-trypsin, IgA, IgM,fibrinogen, α-2-macroglobulin, and complement C3. Of the remaining 10%,about 99% includes: apolipoproteins A1 and B; lipoprotein A; factor H;ceruloplasm; pre-albumin; complement factor B; complement factors C4,C8, C9, and C19; and α-glycoprotein. Often, placing a sorbent such asphenylpropylamine cellulose in the last chamber 102/510 is useful tocatch any remaining biomolecular components in the sample. Generally, ifthe initial sorbent(s) are too general (i.e., have low specificity),then too much material can be sequestered with the first two sorbents,which degrades the usefulness of the remaining sorbents. However, if theinitial sorbents are too specific (i.e., have high specificity), thenthe efficiency of the remaining sorbent materials can be reduced by alarge sample dynamic range. In one embodiment, the sorbents are chosensuch that the first sorbent, or first and second sorbents combined,provide a reduction in the dynamic range of the sample by a factor of atleast 10, more specifically a factor of at least 100, and, still morespecifically a factor of at least 1,000.

The multiplex separation methods of this invention are particularlyuseful for fractionating analytes in complex mixtures, e.g., samplescomprising at least 1000, at least 100,000 or at least 10,000,000different biomolecular species (e.g., proteins). The methods of thisinvention are particularly useful for separating biomolecules frombiological samples. Such samples can include, for example, amnioticfluid, blood, cerebrospinal fluid, intraarticular fluid, intraocularfluid, lymphatic fluid, milk, perspiration plasma, saliva semen, seminalplasma, serum, sputum, synovial fluid, tears, umbilical cord fluid,urine, biopsy homogenate, cell culture fluid, cell extracts, cellhomogenate, conditioned medium, fermentation broth, tissue homogenateand derivatives of these.

An advantage of the present invention is that the device may be usedfor, and is to use and readily adaptable to, any desired number ofseparations. Moreover, other than some basic lab equipment, including,e.g., a peristaltic pump and/or a vacuum station, no additionalequipment may be needed.

In another embodiment the device of this invention is used to apply atwo-dimensional separation method to a mixture of analytes in a sample.Conceptually, this method involves two steps. A first step involvesfractionating analytes into a set of first aliquots based on degree of afirst physical-chemical property using a plurality of first sorbents. Bychoosing appropriate sorbents, the analytes can be partitioned so thateach fraction or aliquot contains analytes falling within a particularrange. A second step involves then fractionating the aliquots (or atleast one aliquot) into a set of second aliquots based on degree of adifferent second physical-chemical property using a plurality ofdifferent second sorbents. Then, the second aliquots can be furtheranalyzed to achieve yet a third dimension of fractionation by, forexample, mass spectrometry. By “physical-chemical property” is meant aproperty of analytes which can be measured and which can serve as thebasis for analyte separation. Preferably, the physical-chemical propertyused in this method is one that varies by degree. For example, proteinsare characterized by isoelectric point, which is one physical-chemicalproperty, and which is measured on a scale from low pI (e.g., pI 3) tohigh pI (e.g., pI 10). As is well known in the art, proteins can beseparated based on isoelectric point using, for example, isoelectricfocusing or ion exchange chromatography. Hydrophobic index is anotherphysical-chemical property that characterized analytes (polypeptideanalytes in particular) and which can be measured on a scale from lowhydrophobic index to high hydrophobic index. As is also well known inthe art, proteins can be separated based on hydrophobic index usinghydrophobic (reverse phase) sorbents or normal phase sorbents. Anotherphysical-chemical property which can be used to fractionate proteins issize, and gel permeation chromatography (size exclusion chromatography)is useful for this purpose. Another physical-chemical property which canbe used to fractionate proteins is metal binding ability. In this case,a series of metal chelate sorbents charged with different metals can beused. Mass is another physical-chemical characteristic of polypeptideanalytes that can be the basis for separation. However, massspectrometry and gel electrophoresis are preferred over chromatographyfor this purpose.

Methods for three-dimensional separation are known in the art. Oneexample is LC-LC-MS. However, one drawback of this method is that ittypically involves sequential elution of analytes from separation media,which can be time consuming. This can involve changing buffer systems toachieve differential elution of analytes from a sorbent. Existingattempts to multiplex this method can involve using multiplechromatography columns in parallel, which can become very costly.However, by combining the device of this invention with chromatographicmedia that use compatible water-based solvent systems, both the time andthe cost impediments can be significantly improved.

A preferred embodiment of this invention involves using pI andhydrophobicity as the two dimensions on which the analytes arechromatographically separated, followed by separation based on massusing mass spectrometry. This method conveniently uses solid buffers forpI separation and hydrophobic sorbents for separation by hydrophobicindex. Solid buffers are described in U.S. provisional patentapplication 60/702,989, filed Jul. 28, 2005 (“Separation of proteinsbased on isoelectric point using solid-phase buffers” Boschetti et al.).They are particularly useful here because they separate according to pIwithout resort to electrophoretic separation or different buffer systemsto achieve step-wise pI fractionation. Any series of reverse phasesorbents can be used. Many of these are commercially available. Anothersorbent that is useful for hydrophobic separation under physiologicalconditions is described in International Publication Number WO2005/073711 (“Chromatographic material for the adsorption of proteins atphysiological ionic strength,” Boschetti et al.) The arrangement ofhydrophobic materials in series from least hydrophobic to mosthydrophobic for the separation of analytes is also described in U.S.provisional patent application 60/591,319, filed Jul. 27, 2004(“Multichemistry fractionation,” Guerrier). All of these documents areincorporated herein by reference.

One method proceeds as follows. A plurality of compartments in one row(or column) of the device of this invention are filled withchromatographic materials that bind analytes based on isoelectric point.For example, a solid phase buffer can be used. The materials arearranged so that the flow of fluid passes the sample mixture through aseries of sorbents that bind the analytes from high to low pI (or,conversely, from low to high pI) and, therefore, from most specific toleast specific. For example, the first sorbent in the series cancapture, and therefore remove, all proteins having a pI above 9. Asecond sorbent can capture all proteins having a pI above 7. Thissorbent will, therefore, partition those proteins the pI of which isbetween 7 and 9. A sorbent in a subsequent compartment in the series cancapture proteins having a pI above 5 and, therefore, will partitionthose proteins whose pI is between 5 and 7. And so on.

In a next step, the fluid conduits of the device are re-routed so thatinstead of connecting compartments in a row series, they now connecteach compartment in the row with compartments in a column. (Conversely,if the first separation is in a column, then the conduits are reroutedinto rows.) A series of compartments in each column is filled with aseries of reverse phase sorbents, each sorbent in the series being morehydrophobic (and, therefore, less specific) than the previous one. Abuffer is now pumped through the conduits so that the analytes capturedon each of the isoelectric sorbents passes sequentially though theseries of hydrophobic sorbents. For example, the hydrophobic sorbentsbuffers in the series can comprise a C₄ chain, a C₈ a chain, C₁₂ chain,C₁₈ chain. This results in a plurality of aliquots, each of which isdefined by a particular pI range and hydrophobic index range.

This method of two-dimensional separation need not be performed entirelyon the device of this invention, or on using the device of thisinvention at all. For example, one can separate analytes in a firstdimension using the device of this invention and then perform theseparation in the second dimension using a different device. In one suchembodiment, after partition of the analytes in a first dimension, theliquid conduits can be removed and the device can be placed on a stackof drip plates or separatable columns so that the outlets of thecompartments align with the wells of the plates or columns. Each well ofa plate in the stack that is aligned under an outlet of the device canbe filled with one of the series of chromatographic materials thatcaptures based on the second separation dimension. Thus, for example,the device of this invention can have compartments arranged into 96 wellformat, having 8 rows, A-H and twelve columns, 1-12. In a firstseparation, the compartments of column 1, e.g. compartments 1A to 1H,can be used to perform the first separation. Then, the device can beplaced on top of a stack of eight filter plates (plates 1-8) also in 96well format, with compartment/well 1A of all the plates alignedvertically under compartment 1A of plate stack. The wells of column 1 ineach plate would contain the same chromatographic material for thesecond separation dimension and they typically would be ordered frommost selective at the top to least selective at the bottom. Elutionbuffer is then pumped through the stack, passing though all wells 1A,all wells 1B, etc. in parallel. The result would be an 8×8 separationset in the wells of the plates. The captured analytes can then be elutedfrom each of these sixty-four wells for further analysis, e.g.,separation of the analytes by mass on, e.g., a mass spectrometer.

Alternatively, the two-dimensional separation just described can beperformed on any device or combination of devices in which thechromatographic media are physically separate and able to separatelysequester the analytes. For example, the method could be performedentirely on drip plates in the 96-well format. In one such embodimentthe first series of chromatographic materials could be placed into well1A of each of eight different filter plates. These plates could bestacked on top of each other in the appropriate order and the samplecould be deposited in the first well on the top of the stack thatcontained the most specific chromatographic material for the firstseparation dimension (for example, a solid buffer with pI 10 furthercomprising an anion exchanger). Then, buffer could be pumped through thecolumn of wells 1A of the eight stacked plates and analytes would beappropriately sequestered according to the first physical-chemicalcharacteristic (in this example, pI). Eluate could be collected from thebottom plate. Now, each of these plates could be independently stackedon top of a stack of filter plates in which well 1A was filled with aseries of chromatographic materials ordered to separate the analytesaccording to the second physical-chemical characteristic. Again, buffercould be pumped through the column of wells 1A, fractionating theanalytes in the second dimension. Then, the analyte contents of all thewells 1A could be eluted and further fractionated, e.g., by mass.

Another device that could be used for this method is a set of stackableand separatable columns. Such columns have compartments in whichchromatographic media is sequestered. The are able to stack on othercolumns by snapping or twisting mechanisms for example.

Proteins can be separated from mixtures based on their pI using a seriesof the chromatographic materials of this invention, each comprising asolid buffer and an ion exchange resin. Each solid buffer comprises anamphoteric macromolecule that confers a predetermined pH to an aqueoussolution. Thus, each chromatographic material produces a particular pH.Proteins passing through, and possessing a pI different from that of,the chromatographic material will have either a net positive charge or anet negative charge or be neutral, depending on whether its pI is,respectively, below, above or the same as the pH of the chromatographicmaterial. Proteins whose charge is opposite that of the ion exchangeresin at the pH of the environment bind to the ion exchanger, whileneutral or same charge proteins remain unbound and pass through thechromatographic material. That is, for example, a protein that isnegatively charged at the pH of the solid buffer will bind to an anionexchange resin. Then, captured proteins can be eluted from thechromatographic material. In one embodiment of the invention, a seriesof chromatographic materials (mixture of solid buffer and ion exchangeresin), each solid buffer producing a different pH, are arranged inseries. Because different proteins are charged at different pH levels,ion exchange resins of each chromatographic material in the seriescaptures a subset of the proteins in a mixture. In this fashion,proteins from biological fluids such as serum, urine, cerebrospinalfluid (CSF), as well as soluble tissue extracts, can be separated as afunction of their isoelectric point.

Chromatographic materials of this invention comprise a solid buffer incombination with an ion exchange resin. In preferred embodiments, eachof these is attached to a solid phase. In particular, this inventioncontemplates a composition comprising solid buffer beads or particlesmixed with ion exchange resin. In another embodiment, solid buffers andion exchange resins are not attached to a solid phase. In anotherembodiment, buffering and ion exchange properties are associated withinthe same particle.

The term “solid buffer” denotes an amphoteric, cross-linked, insolublemacromolecule that is obtained from ionisable monomers, each of whichhas a different pK. A solid buffer confers a predetermined pH to anaqueous solution of diluted electrolytes, and it maintains the pH whenacidic or alkaline molecules are added. In addition to thesephysicochemical properties, a solid buffer useful in this documentcomprises small pores, giving the material a low exclusion limit, forexample lower than 5000 Da.

A low exclusion limit prevents proteins from diffusing inside thecross-linked monomers while maintaining full diffusion for small ions.Such restricted diffusion minimizes the risk of non-specific binding tothe solid buffer. In some embodiments, a solid buffer comprises anexclusion limit lower than 5000 Da, 4000 Da, or 3000 Da. Amphotericmacromolecules with small pores can be generated by using relativelyconcentrated monomer solutions and or high degrees of crosslinkingmonomers.

In one aspect, a solid buffer can be a polymer. For example, a solidbuffer can be a polyacrylamide or a block copolymer. In another example,solid buffers can be made by combining acrylamide monomers of differentpK to reach buffering power around at a predetermined pH.

Solid buffers can be prepared using routine chemicals and methods usedin the arts of polymer chemistry and biochemistry. In general, to createa solid buffer for a particular pH, a monomer with a corresponding pK isselected (at a concentration that can range from few mM to severalhundred mM) and is titrated to a pH close or same to its pK with acomplementary monomer. For example, if the selected monomer has a pK of8.0 it will be titrated to pH close to 8.0 using a monomer of a pK lowerthan 4.5. If a monomer of the desired pK is not available, a mixture ofmonomers (pK above and below the desired pK) can be used, followed bytitration to a pH between the two pKs. Appropriate cross-linkingreagents and polymerization catalysts are then added to the pH-adjustedsolution of monomers in proportions sufficient to cause polymerizationto generate particles of the solid buffer.

A variety of monomers are commercially available. Exemplarily monomersinclude, but are not limited to, N-acryloylglycine, 4-acrylamidobutyrricacid, 2-morpholino-ethylacrylamide, 3-morpholinopropylacrylamide,N,N-dimethylaminoethylacrylamide and N,N-dimethylaminopropylacrylamide.Immobilines also can be used to make solid buffers.

Immobilines are acrylamide derivatives that conform to the generalformula:

where R includes a group that provides the characteristic pI. See, e.g.,U.S. Pat. No. 4,971,670. While this characterization in principleembraces many molecules, Amersham produces molecules, marketed under thetrademark IMMOBILINE®, that are particularly suited for creatingisoelectric gels and polymers. The IMMOBILINE® collection of moleculesincludes the following, having the pI indicated in parenthetical:N-acryloylglycine (pK 3.6); 4-acrylamidobutyrric acid (pK 4.6);2-morpholinoethyl-acrylamide (pK 6.2); 3-morpholinopropylacrylamide (pK7.0); N,N-dimethylamino-ethylacrylamide (pK 8.5); andN,N-dimethylaminopropylacrylamide (pK 9.3) (collectively, “theimmobilines”). Any of the immobilines can be combined, as monomers, andco-polymerized with acrylamide and N,N′-methylenebisacrylamide oranother suitable cross-linking agent, to produce a desired pI specificpolymer. Acrylamide can be substituted by other non-ionic acrylamidederivatives, such as N-isopropylacrylamide, methylacrylamide,methylolacrylamide dimethylacrylamide, diethylacrylamide,tris(hydroxymethyl)methylacrylamide, etc.

A variety of resources are available to assist in the selection ofcombinations and concentrations of monomers to produce a solid bufferwith a particular pH. For example, formula tables for monomercombinations are provided in JOURNAL OF CHROMATOGRAPHIC LIBRARY, VOLUME63 Chapter 12 (Righetti, Stoyanov & Zhukov eds., 2001). AmershamBiosciences provides similar formula tables in its “Protocol Guide #1:Isoelectric Membrane Formulas for IsoPrime Purification of Proteins.”Seewww4.amershambioscieces.com/aptrixupp00919.nsf/(FileDownload)?OpenAgent&docid=FD3302088BD37BC6C1256EB400417E5C&file=80635018.pdf.

Algorithms for selecting concentrations of monomers are available, too.See, e.g., Giaffreda et al., J. Chromatog., 630:313-327 (1993). Inaddition, techniques for determining the pI for polymers are well-knownin the art. Examples of such methods include Ribeiro et al., Computersin Biology & Medicine 20: 235-42 (1990), Ribeiro et al., loc. cit., 21:131-41 (1991), and Sillero et al., Analytical Biochemistry 179: 319-25(1989).

In one embodiment, the particles described in International ApplicationPCT/US2005/007762, which is hereby incorporated by reference, can beused as solid buffers.

In another embodiment, a solid buffer is polymerized within cavities ofa solid support. In other embodiments, the solid buffer is deposited onthe interior and exterior surfaces of a solid support, such as theinterior and exterior surfaces formed by the interior pore volume ofcavities in a particle. The deposition can be by chemical bond or othermeans.

Amino acids and peptides can provide such surface layers, as they havedefined isoelectric points. Thus, in some embodiments, a solid buffercomprises two or more amino acids. The amino acids can be any of thetwenty naturally-occurring amino acids, or the amino acids can besynthetic amino acids. Useful amino acids include those among the twentynaturally occurring amino acids having ionizable side chains, including:lysine, arginine, glutamic acid, aspartic acid, serine, cysteine,threonine, tyrosine, asparagines, glutamine. In addition, it will beunderstood by those in the art that other compounds having defined pIvalues that can be attached to the interior and exterior particlesurfaces as described above can be used with the present invention.Linkers can be used to provide attachment sites on the surface of aparticle.

In general, a solid buffer should not be able to adsorb proteins byitself, should have the same density as the ion exchanger, and shouldhave a good buffering capacity at the desired pH.

The term “solid support” denotes a solid, porous material wherein ionexchange polymers or solid buffers can be attached or loaded to preventpolymer collapse under low concentration.

Chromatographic material can utilize a variety of solid supports.Illustrative of solid supports in this context are particles, membranes,and monoliths. A “monolith” is a single piece of material, generallyporous, to which chromatographic ligands can be attached. Generallymonoliths have significantly greater volume than beads, for example, inexcess of 0.5 mL per cm³ of monolith.

In one embodiment, the solid support comprises a substantially porousparticle having a plurality of cavities extending inwardly from thesurface. The particles preferably have sizes, mechanical strengths andbuoyancies that are compatible with separating biological extracts. Inone embodiment, the particles comprise one or more mineral oxides suchas silica, titania, zirconia, hafnia, alumina, gallia, scandia, yttria,actinia, or a rare earth mineral oxides.

When porous particles are employed as solid supports for solid buffers,relatively large particles can be used to minimize the external surfacearea relative to the particle volume and hence reduce the risk ofnon-specific binding for proteins. In some embodiments, the particleshave diameters greater than about 50 μm, or greater than about 100 μm,or greater than about 200 μm. In one example, particles of about 150 μmare used as solid supports for solid buffers. Pore volume of theparticle can range from 10 to 70% of the overall particle volume.

Alternatively, a porous, plastic bead can serve as solid support.Polystyrene is a well-known polymer that can be formed into beads havingpores, for chromatography. Other synthetic polymers also can be used;for instance, those based on acrylics, such as methymethacrylates, aswell as porous nylons, porous polyvinyl plastics, and polycarbonates.Additional examples of porous particle bodies are described in U.S. Pat.Nos. 6,613,234, 5,470,463, 5,393,430 and 5,445,732, each of which isincorporated herein by reference.

Ion exchange chromatography separates compounds based on their netcharges. Negatively or positively charged functional groups arecovalently bound to a solid support matrix, yielding either a cation oranion exchanger, respectively. When a charged compound is applied to anexchanger of opposite charge, it is adsorbed, while compounds that areneutral or the same charge are eluted in the void volume of the column.Binding of the charged compounds is reversible, and adsorbed compoundsare commonly eluted with a salt or a pH gradient.

The term “ion exchange resin” refers to a solid, porous network (mineralor organic or composite) carrying ionizable groups of positive ornegative sign and of a single group. Positively charged ionic groups(anion exchangers) are, for example, quaternary, tertiary and secondaryamines and pyridine derivatives. Negatively charged ionic groups (cationexchangers) are, for example, sulfonates, carboxylates and phosphates.

Selection of an ion exchange resins depends on the properties of thecompounds to be separated. For amphoteric compounds, the pI of thecompound and its stability at various pH values determine the separationstrategy. At a pH above its pI, the compound of interest will benegatively charged, and at a pH below its pI the compound will bepositively charged. Thus, if the compound is stable at a pH above itspI, an anion exchange resin is used. Conversely, if the compound isstable at a pH below its pI, a cation exchange resin is used. Theoperating pH also determines the type of exchanger to use. A strong ionexchange resin maintains capacity over a wide pH range, while a weak oneloses capacity when the pH no longer matches the pK_(a) of itsfunctional group.

Anion exchangers can be classified as either weak or strong. The chargegroup on a weak anion exchanger is a weak base, which becomesdeprotonated and, therefore, loses its charge at high pH. DEAE-celluloseis an example of a weak anion exchanger, where the amino group can bepositively charged below pH ˜9 and gradually loses its charge at higherpH values. A strong anion exchanger, on the other hand, contains astrong base such as a quaternary amine, which remains positively chargedthroughout the pH range normally used for ion exchange chromatography(pH 2-12).

Cation exchangers also can be classified as either weak or strong. Astrong cation exchanger contains a strong acid (such as a sulfopropylgroup) that remains charged from pH 1-14; whereas a weak cationexchanger contains a weak acid (such as a carboxymethyl group), whichgradually loses its charge as the pH decreases below 4 or 4.5.

In one embodiment of the invention, strong ion exchangers such asquaternary amines or sulfonic acids are used. Weak ion exchangers, suchas tertiary amines and carboxylic acids, also can be used, for example,when separating proteins that have pIs between 5 and 8.

Table 4 provides a list of common ion exchangers.

TABLE 4 Strong Anion CH₂N⁺(CH₃)₃ Triethylaminomethyl C₂H₄N⁺(C₂H₅)₃Triethylaminoethyl C₂H₄N⁺(C₂H5)₂CH₂CH(OH)CH₃ Diethyl-2-hydroxypropylaminoethyl R₁R₂R₃R₄N⁺ Quaternary amine Weak Anion C₂H₄N⁺H₃Aminoethyl C₂H₄NH(C₂H₅)₂ Diethylaminoethyl CH₂ CH₂N(C₂H₅)₂(—CH₂CH₂—NH(C₂H₅)₂) DEAE-cellulose Or CH₂ CH₂NH(C₂H₅)₂ Strong Cation SO₃ ⁻Sulpho CH₂SO₃ ⁻ Sulphomethyl C₃H₆SO₃ ⁻ Sulphopropyl CH₃SO₃ ⁻Methylsulfonate Weak Cation COO⁻ Carboxy CH₂COO⁻ Carboxymethyl

Ion exchange resins are well known in the art. Commercially availableion exchangers useful in this invention include, but are not limited to,Q HyperD, S HyperD, Q Sepharose, S Sepharose, Q HyperZ and CM HyperZ.These resins can be mixed with solid buffer beads, for example, toproduce the chromatographic materials of this invention.

Chromatographic material can comprise a volume of ion exchanger from 5%to 95% with the remainder being solid buffer.

Chromatographic material useful for separating proteins from mixturesbased on their pI can be produced by combining a solid buffer with anion exchange resin. In one embodiment, a solid buffer and ion exchangeresin, each attached to different solid supports, are combined to form amixture. Moreover, the mixture may be a bed of mixed particles.

In another embodiment, a chromatographic material comprises a solidbuffer and an ion exchange resin attached to a single solid support,such as a membrane or monolith. In another example, an ion exchangeresin is combined with a solid buffer on a single particle. In thisembodiment, a solid buffer attached to a particle is prepared first,then a polymer with ion exchange properties and large pores is formed ontop of the solid buffer.

Proteins passing through chromatographic material of the invention(e.g., through a column or through a series of chambers 102) will haveeither a net positive charge or a net negative charge or be neutral,depending on whether their pIs are, respectively, below, above or thesame as the pH generated by the solid buffer. Proteins whose charge isopposite that of the ion exchange resin at the pH of the environmentbind to the ion exchanger, while neutral or same charge proteins remainunbound and pass through the chromatographic material. That is, forexample, a protein that is negatively charged at the pH of the solidbuffer will bind to the ion exchange resin if this latter is an anionexchanger. Then, captured proteins can be eluted from thechromatographic material. Thus, the chromatographic material of theinvention are useful for separating proteins based on pI.

In another embodiment, proteins are separated from mixtures based ontheir respective pI using a series of chromatographic material. In amulti-staged column (or series of chambers 102) comprised of differentchromatographic material, a discontinuous gradient of pH is generated.Proteins in a mixture passing through such a column (or series ofchambers 102) will become ionized differently according to theirlocation in the column (or series of chambers 102). When a proteinobtains a charge opposite that of the ion exchange resin, it will bindto it. Thus, a mixture traveling through the column (or series ofchambers 102) will be depleted of one protein category at a time as itcrosses the different sections of the column (or series of chambers102).

Thus, one of the separation devices shown in FIGS. 1-10 can comprise aseries of chromatographic materials that are successively placed in thechambers 102 of the device. The series of chromatographic materials mayinclude a cation exchanger and solid buffers of pH 9, 7 and 5,respectively (from the top to the bottom). A sample of a biologicalextract is applied first to a container holding the solid buffer of 9.Proteins that have a pI above pH 9 will be positively charged in thisenvironment and, therefore, will bind to the ion exchanger part of thechromatographic material in a first of the chambers 102; in mostbiological extracts this represents a minority of the protein species.The neutral or negatively charged proteins will not be bound and areeluted from the first chamber 102/510. This eluate is then loaded to asecond chamber 102/510 holding the solid buffer of pH 7. At this pH, theproteins having a pI above 7 will be positively charged and will bind tothe ion exchanger. The neutral and positively charged proteins areunbound and are eluted from the second chamber 102/510 holding thechromatographic material. Accordingly, this section of chromatographicmaterial has captured proteins having a pI between 7 and 9, with theproteins that have a pI above 9 having already been captured in thefirst chamber 102/510. Then, this second eluate is loaded to a thirdchamber 102/510 holding chromatographic material with solid buffer of pH5. According to a similar mechanism described above, proteins of pIbetween 5 and 7 will be captured by the cation exchanger, proteins withpI at or below 5 will not be captured and will be found in the eluate.The bound proteins, which define sub-samples, can be eluted thereafter,by conventional means, from the various chambers 102. Accordingly, theproteins have been fractionated in sub-samples having pI above 9, pIbetween 7 and 9, pI between 5 and 7 and pI below 5. In a similar way,proteins can be fractionated by cation exchange chromatography, usingchromatographic materials composed of solid buffers of increasing pH.

In one aspect, the series can comprise two, three, four, five, six,seven, eight, nine, ten, eleven or twelve different chromatographicmaterials. As described above, the chromatographic materials arearranged in the column or chambers 102 in order of increasing ordecreasing pH depending on whether an anion exchanger or a cationexchanger, respectively, is used.

In preparing a solution for loading onto a chromatographic adorbent,electrolytes such as simple salts, for example, sodium chloride orpotassium chloride, can be used. As biological molecules can act aselectrolytes, however, neat water can be used. Modifiers also can beadded to a mixture to prevent proteins from aggregating. Examples ofsuch modifiers include, but are not limited to, glycols and non ionicchaotropic agents, such as urea or non-ionic detergents.

Bound proteins can be desorbed using any chemical component capable ofeluting proteins from an ion exchange resin. Most generally, saltsolutions are used to desorb proteins; however, a pH change also can beused, as well as displacers.

In another embodiment, there is provided an apparatus comprising aseries of containers, wherein a first container in the series comprisesa fluid inlet and a last container in the series comprises a fluidoutlet, and each container in the series is in fluid communication witha next container in the series, and wherein each container in the seriescomprises a different chromatographic material and the containers arearranged in increasing or decreasing order according to the pH of thechromatographic materials.

In one such embodiment, the chromatographic materials are containedwithin cartridge or container segments having inlets and outlets. Thesegments are stackable with and detachable from each other. The segmentseach contain a different chromatographic material. They can containfilters or membranes that hold the chromatographic material in place.When attached end-to-end, the segments create a column into which asolution can be poured. After the fluid has passed through all of thesegments, the segments can be detached from one another and the capturedproteins eluted from each segment. See, for example, WO 03/036304(Schultz et al.).

In another such embodiment, the container is a well of a microtiterplate such as that shown in FIGS. 1-3; each container in the series isdefined by a chamber 102/510 that is connected by removable conduits. Aspreviously discussed, the plate can be a drip plate or a filter plate.In other embodiments, however, the plate can comprise a piece comprisingchannels, such as bores, that open on either side of the piece and thatwill define chambers when conduits are attached to the openings of thebores. Preferably, the chambers 102 are arrayed substantially in aplane.

It is understood that the following examples are for illustrativepurposes only and that various modifications or changes in light thereofwill be suggested to persons skilled in the art and are to be includedwithin the spirit and purview of this application and scope of theappended claims.

Example 2 Preparation of Solid Buffer of pH 8.5

A solid-phase buffer of pH 8.5 can be prepared by dissolving 10 mMolesof N,N-dimethyl-aminopropyl-acrylamide having a pK of 8.5 (this is 1420mg of free base) in 50 mL of water. The solution is titrated to pH 8.5by slow addition of the monomer acrylamidoglycolic acid of pK 3.1 (freeacid). Next, 30 g of acrylamide and 2 g of methylene-bis-acrylamide areadded to the solution. The volume of the solution is then raised to 100mL. To this final solution, polymerization catalysts are added, forexample ammonium persulfate and TEMED. The solution is then used, forexample, to impregnate porous particles. Once the hydrogel haspolymerized inside the pores of the particles, the material is washed toremove by-products and reagent excess. The solid-phase buffer can bestored in the presence of 20% ethanol.

Example 3 Preparation of Solid Buffer of pH 4.6

A solid-phase buffer of pH 4.6 can be prepared by dissolving 100 mM ofN-acryloyl glycine (pK 4.6) in 1 liter of distilled water. The solutionis then titrated to pH 4.6 by adding a concentrated solution (50% inwater) of N,N,N-triethyl aminoethyl acrylamide. To the obtainedsolution, 200 gram of acrylamide and 10 grams ofN,N′-methylene-bis-acrylamide are added. The mixture is stirred tocomplete solubilization. A catalysis system composed ofN,N,N′,N′-tetramethylethylene diamine and ammonium persulfate is addedto the solution just before use. 38 mL of the final solution is added to100 mL of porous zirconia beads of about 75 μM (pore volume of 38 mL for100 gram) and mixed to the complete absorption of the solution in theporous volume of the mineral beads. Next, the impregnated beads aredegassed under vacuum three times, alternating the introduction ofnitrogen. The mixture is then stored at room temperature in the presenceof nitrogen until polymerization of the monomers. The resulting solidbuffer is washed extensively with water, with a buffer of the same pH(e.g., acetate buffer pH 4.6), and again with water. The solid buffer isthen ready for use in the presence of an ion exchanger.

Example 4 Preparation of Solid Buffer of pH 9.3

A solid buffer of pH 9.3 can be prepared by dissolving 100 mM ofN,N-dimethyl aminopropyl acrylamide (pK 9.3) in 1 liter of distilledwater. The solution is then titrated to pH 9.3 by adding a concentratedsolution (50% in water) of acrylic acid. To the obtained solution, 200gram of dimethyl-acrylamide and 10 grams ofN,N′-methylene-bis-acrylamide are added. The resulting mixture isstirred to complete solubilization. A catalysis system composed ofN,N,N′,N′-tetramethylethylene diamine and ammonium persulfate is addedjust before use. 38 mL of the final solution is added to 100 mL ofporous zirconia beads of about 75 μM (pore volume of 38 mL for 100 gram)and mixed to complete absorption of the solution in the porous volume ofthe beads. Next, the impregnated beads are degassed under vacuum threetimes, alternating the introduction of nitrogen. The mixture is thenstored at room temperature in the presence of nitrogen untilpolymerization of monomers. The resulting material is washed extensivelywith water, a buffer of the same pH (e.g., Tris-HCl buffer, pH 9.3), andagain with water. The solid buffer is then ready for use in the presenceof an ion exchanger.

Example 5 Preparation of Solid Buffer of pH 7.7

A solid buffer of pH 7.7 can be prepared by dissolving 50 mM of3-morpholinopropyl acrylamide (pK 7.0) and 50 mM ofN,N-dimethylaminoethyl acrylamide (pK 8.5) in 1 liter of distilledwater. The solution is then titrated to pH 7.7 by adding a concentratedsolution (50% in water) of 2-acrylamido-2-methylpropane sulfonic acid.To the obtained solution, 200 gram of acrylamide and 10 grams ofN,N′-methylene-bis-acrylamide are added, and the resulting mixture isstirred to complete solubilization. A catalysis system composed ofN,N,N′,N′-tetramethylethylene diamine and ammonium persulfate is addedjust before use. 38 mL of the final solution is added to 100 mL ofporous zirconia beads of about 75 μM (pore volume of 38 mL for 100 gram)and mixed to the complete absorption of the solution in the porousvolume of the beads. Next, the impregnated beads are degassed undervacuum three times, alternating the introduction of nitrogen. Themixture is then stored at room temperature in the presence of nitrogenuntil polymerization of monomers. The resulting material is washedextensively with water, a buffer of the same pH (e.g., morpholine-HClbuffer pH 7.7), and again with water. The solid buffer is then ready foruse in the presence of an ion exchanger.

Example 6 Preparation of a Mix Mode Chromatographic Material SolidBuffer of pH 4.6 and Cation Exchanger

A solid buffer of pH 4.6 can be prepared by dissolving 150 mM ofN-acryloyl glycine (pK 4.6) and 10 mM N,N′-methylene-bis-acrylamide in 1liter of distilled water. The solution is then titrated to pH 4.6 byadding a concentrated solution (50% in water) of N,N,N-triethylaminoethyl acrylamide. A catalysis system composed ofN,N,N′,N′-tetramethylethylene diamine and ammonium persulfate is addedjust before use.

Separately a second aqueous solution composed of 5% of2-acrylamido-2-methylpropane sulfonic acid sodium salt, 5% ofdimethylacrylamide and 1% of and mM N,N′-methylene-bis-acrylamide isprepared. The pH of this solution is then adjusted to 4.6 by addition ofa base or an acid. A catalysis system composed ofN,N,N′,N′-tetramethylethylene diamine and ammonium persulfate is addedjust before use.

100 mL of porous zirconia beads of about 75 μM (pore volume of 40 mL for100 gram) are mixed with 20 mL of the first monomer solution and thentreated as described in the previous examples up to the polymerization.This intermediate product is washed extensively with water, with abuffer of the same pH (e.g., acetate buffer pH 4.6), and again withwater. The washed product is then dried using, e.g., repeated washeswith dry ethanol and acetone.

The intermediate dry product is then mixed with 20 mL of the secondmonomer solution (to fill up the total porous volume of the mineralbeads). A second polymerization process is then started as above. Thefinal chromatographic material is washed extensively with water, withacetate buffer pH 4.6, and with distilled water.

Example 7 Preparation of Solid Buffer of pH 6.5

A solid buffer of pH 6.5 can be prepared by dissolving 150 mM of3-morpholinopropyl acrylamide (pK 7.0) in 1 liter of distilled water.The solution is then titrated to pH 6.5 by adding a concentratedsolution (50% in water) of N-acryloyl glycine (pK 3.6). To the obtainedsolution 400 gram of acrylamide and 30 grams ofN,N′-methylene-bis-acrylamide are added, and the resulting mixture isstirred to complete solubilization. A catalysis system composed ofN,N,N′,N′-tetramethylethylene diamine and ammonium persulfate is addedjust before use. 100 mL of this solution is dispersed in 500 mL ofparaffine oil containing 3% of arlacel C (oil-soluble emulsifier). Thesuspension is maintained under stirring for 3 hours at 65° C. to allowmonomers to copolymerize together. Hydrogel beads formed duringpolymerization are collected by filtration and washed extensively with anon-polar solvent to eliminate traces of paraffin oil. Next, the beadsare washed extensively with water, with a buffer of the same pH, andagain with water. The solid buffer is then ready for use in the presenceof an ion exchanger.

Example 8 Preparation of Solid Buffer of pH 9.0 Using IrregularParticles

A solid buffer of pH 6.5 can be prepared by dissolving 150 mM ofN,N,N-triethyl-aminoethyl-acrylamide (pK 12) in 1 liter of distilledwater. The solution is then titrated to pH 9.0 by adding a concentratedsolution (50% in water) of acrylamidoglycolic acid (pK 3.1). To theobtained solution, 300 gram of dimethyl-acrylamide and 20 grams ofN,N′-methylene-bis-acrylamide are added, and the resulting mixture isstirred to complete solubilization. A catalysis system composed ofN,N,N′,N′-tetramethylethylene diamine and ammonium persulfate is addedjust before use. The solution then is placed in a warm bath of 65° C.under nitrogen. Five hours later the polymerization is complete under afull hydrogel block. The hydrogel is cut into small pieces and ground toget particles of about 100 μm. Particulated product is then washedextensively with water, then with a buffer of the same pH, and againwith water. The solid buffer is then ready for use in the presence of aion exchanger.

Example 9 Separation of Proteins Based on pI Using an Anion ExchangerMixed with Three Different Solid Buffers

A series of three chromatographic materials are assembled in a series ofinterconnected sectional columns or chamber 102/510 (outlet of theprevious to the inlet of the following) column or chamber 102/510. Eachof the chromatographic materials comprises the same anion exchanger (QHyperZ sorbent) but a different solid buffer of different pH, inparticular, 5.4, 7.7 and 9.5, respectively. The chromatographicsectional columns or chambers 102 are aligned in order of increasing pH,i.e., the first sectional column or chamber 102/510 has a pH of 5.4 andthe last adsorbent has pH of 9.5.

The Q anionic sorbent is positively charged in all pH ranges induced bythe solid-phase buffers (5.4 to 9.5).

A sample containing five proteins (lysozyme (pI 11), cytochrome c (pI9.0), myoglobin (pI 7.0), human albumin (pI 6.0) and fetuin (pI<5.0)) isprepared in 10 mM potassium chloride. To avoid protein-proteininteraction, 2M urea is added. The sample is then loaded onto the columnor into a first chamber 102/510.

At the first chromatographic material, only fetuin adsorbs because it isthe only protein negatively charged at pH 5.4. The remaining fourproteins will progress to the second chromatographic material in thecolumn or in a second chamber 102/510.

At the second chromatographic material, both albumin and myoglobin arenegatively charged at pH 5.4. Thus, they are captured by the anionexchanger.

At the third chromatographic material in the column or in a thirdchamber 102/510, only cytochrome C adsorbs because it is the onlyprotein remaining in the sample that is negatively charged at pH 9.5.

Lysozyme possesses a net positive charge at pH 9.5. Accordingly, it willexit the column or the third chamber 102/510 with the flow-through.

Once the adsorption phase is over, the chromatographic materials aredisconnected and separately treated to desorb proteins. The boundproteins are desorbed using 1 M potassium chloride, thereby yielding aplurality of sub-samples.

Example 10 Separation of Proteins Based on pI Using a Cation ExchangerMixed with Three Different Solid Buffers

A series of three chromatographic materials are assembled in a series ofinterconnected sectional columns or chambers 102 (outlet of the previousto the inlet of the following) column. Each of the chromatographicmaterials comprises the same cation exchanger but a different solidbuffer of different pH, in particular, 5.4, 7.7 and 9.5, respectively.The chromatographic sectional columns or chambers 102 are aligned inorder of decreasing pH, i.e., the first sectional column or chamber102/510 has a pH of 9.5 and the last adsorbent has a pH of 5.4.

The cation exchanger is negatively charged in all pH ranges induced bythe solid-phase buffers (5.4 to 9.5).

A sample containing five proteins (lysozyme (pI 11), cytochrome c (pI9.0), myoglobin (pI 7.0), human albumin (pI 6.0) and fetuin (pI<5.0)) isprepared in 10 mM potassium chloride. To prevent protein-proteininteraction, 2M urea is added. The sample is then loaded onto the columnor into a first chamber 102/510.

At the first chromatographic material, only lysozyme adsorbs because itis the only protein negatively charged at pH 9.5. The remaining fourproteins progress to the second chromatographic material in the chamberor to a second chamber 102/510.

At the second chromatographic material, only cytochrome C adsorbsbecause it is the only protein remaining in the sample that isnegatively charged at pH 7.7. The remaining proteins progress to thethird chromatographic material in the column or to a third chamber102/510.

At the third chromatographic material, both albumin and myoglobin arenegatively charged at pH 5.4. Thus, these proteins are captured by theanion exchanger.

Fetuin possesses a net positive charge at pH 5.4. Thus, fetuin will exitthe column or the third chamber 102/510 with the flow-through.

Once the adsorption phase is over, the chromatographic materials aredisconnected and separately treated to desorb proteins. The boundproteins are desorbed using 1 M potassium chloride, thereby yielding aplurality of sub-samples.

IV. Fractionation Based on Hydrophobic Index

If there are other methods of separating according to hydrophobic indexbesides the one we describe here, they should be mentioned.

Another embodiment of the present invention provides methods and systemsfor reducing the complexity of complex mixtures containing biomolecularcomponents (i.e., chemical species generated by biological processessuch as proteins, nucleic acids, lipids, metabolites, etc.) by isolatingand detecting biomolecular components, while achieving greatersensitivity and efficiency than heretofore possible.

FIG. 12 provides an illustration of one embodiment of the invention at1000. A sample solution containing a complex mixture including aplurality of different biomolecular components 1001 is introduced to asample fractionation column 1002 (or series of chambers 102/510) for atleast partial resolution as described hereinbelow. The column 1002 (orseries of chambers 102/510) includes a plurality of sorbent materials1004, 1006, 1008, and 1010 arranged serially (e.g., in successivechambers 102/510) and through which the sample solution 1001 issuccessively passed, thereby isolating each of the sorbent materials andenabling any remaining solution to be eluted to a receptacle 1012.

In one embodiment of the invention, the sorbent materials are chosensuch that substantially all of the biomolecular components are capturedby sorbents 1004, 1006, 1008, 1010. In a more particular embodiment ofthe present invention, each of the sorbents 1004, 1006, 1008, 1010captures a substantially unique subset of the plurality of biomolecularcomponents. Thus, sorbent 1004 is effective to capture sub-sample 1014,sorbent 1006 is effective to capture sub-sample 1016, sorbent 1008 iseffective to capture sub-sample 1018, and sorbent 1010 is effective tocapture sub-sample 1020. Following capture of the various sub-samples ofthe plurality of biomolecular components in the sample solution 1001,the sorbents, including the captured biomolecular components, may bemore permanently isolated (i.e., removed from the column or chambers102/510). The sub-samples may be eluted or otherwise removed from thesorbents for further processing pursuant to a multidimensionalseparation protocol, as later discussed in greater detail.

The multiplex separation methods of this invention are particularlyuseful for fractionating analytes in complex mixtures, e.g., samplescomprising at least 1000, at least 100,000 or at least 10,000,000different biomolecular species (e.g., proteins). The methods of thisinvention are particularly useful for separating biomolecules frombiological samples. Such samples can include, for example, amnioticfluid, blood, cerebrospinal fluid, intraarticular fluid, intraocularfluid, lymphatic fluid, milk, perspiration plasma, saliva semen, seminalplasma, serum, sputum, synovial fluid, tears, umbilical cord fluid,urine, biopsy homogenate, cell culture fluid, cell extracts, cellhomogenate, conditioned medium, fermentation broth, tissue homogenateand derivatives of these.

An advantage of the present invention is that the device is to use andreadily adaptable to any desired number of separations. Moreover, otherthan some basic lab equipment, including, e.g., a peristaltic pumpand/or a vacuum station, no additional equipment may be needed.

In another embodiment, this invention provides a kit. The kit contains adevice of this invention and at least one container comprising aseparation medium. Such a kit is useful because it enables the user todefine the particular arrangement of chambers that will comprise theseparation media and what the particular series of separation media willbe. Accordingly, in some embodiments, the kit comprises a plurality ofdifferent separation media. The kit optionally can comprise at least onecontainer that comprises a fluid, such as a buffer, for use intransporting a sample from one chamber to another during the use of thedevice.

Example

A separation protocol of human serum using one of the aforementionedembodiments is hereafter set forth.

Step 1: Chambers within a column of a 96-chamber (eight by twelve)microtiter filter plate (Princeton Separations, NJ, USA, long dripnozzles, 25 μM membrane pore size) of the type shown in FIG. 10 werefilled with 150 μl (total packed bed volume) of the followingfunctionalize resins: protein-A, blue trisacryl, MEP, heparin, green dyeligand and phenylpropyl.

Step 2: After connecting (a) the outlet of the first chamber to theinlet of the second chamber, (b) the outlet of the second chamber toinlet of the third chamber (and so on) with plastic tubing, aperistaltic pump (Bio-Rad model EP-1 Econo Pump) was connected to theinlet of the first chamber. Twenty volumes of binding buffer (1×PBS pH7[16 volumes], 1 MTris-HCl buffer pH 8 [9 volumes] and deionized water[75 volumes]) were then pumped through the chambers (i.e., across theseries of resins) at a flow rate of 0.2 ml/min.

Step 3: After equalization with the binding buffer volumes, 40 μl ofdenatured serum (human serum from Intergen, Norcross, Ga., USA, whichwas denatured by adding 2 ml serum to 2.5 mL 9M Urea, 2% CHAPS(3-[(cholamidopropyl)dimethylamino]-1-propanesulfonate) for one hour atroom temperature) was diluted 1:5 in the binding buffer. The dilutedserum was then introduced into the inlet of the first chamber.

Step 4: The flow through the chambers was reestablished at a rate of0.02 ml/min using the binding buffer. For the first 60 minutes, theeffluent from the outlet of the final chamber was discarded.

Step 5: After the first 60 minutes, the effluent was collected for anadditional 60 minutes.

Step 6: After a total of 120 minutes, the flow was stopped and allplastic tubing was removed from the microtiter plate.

Step 7: Excess buffer within each of the wells was removed by vacuum anddiscarded.

Step 8: Captured proteins from each of the wells were then individuallyeluted using either (i) TFA (0.8 volumes)-water (79.2 volumes)-ACN (6.6volumes)-IPA (13.4 volumes) or (ii) ammonium hydroxide (8 volumes)-water(72 volumes)-ACN (6.6 volumes)-IPA (13.4 volumes). Specifically, proteinelution was performed by incubating the sorbents while gently mixing for20 minutes.

Step 9: Supernatants from each sorbent were recovered by vacuumfiltration into a collection plate, frozen, and lyophilized. Lyophilizedresidue was then re-dissolved in 100 μL of 25 mM Tris-HCl buffer, pH 7.5or 150 μl 50 mM Hepes, 0.1% OGP buffer. Aliquots of the re-dissolvedlyophilized residue were then analyzed by SELDI-mass spectrometryanalysis (Ciphergen Biosystems Inc., Fremont, Calif., USA). Using thismethod, a total of eight fractions, including flow-through, wereobtained from each sample.

Step 10: Each spot of a Q10 ProteinChip array (Ciphergen BiosystemsInc.) was equilibrated two times with 150 μL of the indicatedarray-specific binding buffer for five minutes as described completelyin the manufacturer's instructions. Each spot surface was then loadedwith 30 μL (50 μL in case of 150 μL of dissolving buffer) of the samplepreviously half-diluted in the array binding buffer.

Step 11: After an incubation period of 30 minutes under vigorousshaking, each spot was washed two times with 150 μL of the bindingbuffer for five minutes to eliminate non-adsorbed proteins.Subsequently, each spot was quickly rinsed with deionized water.

Step 12: All surfaces were dried and loaded twice with 0.5 μL of asaturated solution of SPA in a mixture of ACN (49.5 volumes)-TFA (0.5volumes)-deionized water (50 volumes), and dried again.

Step 13: All arrays were then analyzed using a mass spectrometer readerused in a positive ion mode, with an ion acceleration potential of 20 kVand a detector voltage of 2.8 kV. The molecular mass range investigatedby mass spectrometer m/z was from 0 to 300. Focus mass was set at 30 and7 for high- and low-mass range, respectively. Laser intensityresponsible for the desorption/ionization of proteins on the spotsurface was set at 200 and 180 units for high- and low-mass range,respectively, and sensitivity of the detector at 9 units.

Step 14: The results of the mass spectrometry are shown in FIG. 11,which charts the fractionation of the human serum.

Although the aforementioned describes embodiments of the invention, theinvention is not so restricted. It will be apparent to those skilled inthe art that various modifications and variations can be made to thedisclosed embodiments of the present invention without departing fromthe scope or spirit of the invention.

For example, the aforementioned tubes/conduits could be replaced and/orenhanced with a series of valves, as shown in FIGS. 12A-12F.Specifically, each chamber in a standard 96-well plate could beconnected to each of the three or four chambers immediate adjacentthereto in the same row and column by conduits in which valves arepresent, as shown in FIG. 12A. During a first separation protocol (e.g.,pI separation) shown in FIG. 12B, the valves connecting each of thechambers along a particular row could be opened whereas all other valvesare closed. As a result, a first separation protocol could proceedthrough the valve-open conduits in the row. Subsequently, each of thevalves along the row could be closed followed by an opening of each ofthe valves in one or more of the columns intersecting the row, as shownin FIG. 12C. Second separation protocols (e.g., hydrophobicityseparation) could then proceed through the valve-open conduits in thecolumns, as shown in FIG. 12D. Further, if the second separationprotocol in FIG. 12D proceeded through only one column, each of thevalves along that column could be closed followed by an opening of eachof the valves in one or more of the other rows intersecting the column,as shown in FIG. 12E. Third separation protocols (e.g., massspectrometry) could then proceed through the valve-open conduits inthose rows, as shown in FIG. 12F.

Accordingly, these other fluidic devices and related separation methodsare fully within the scope of the claimed invention. Therefore, itshould be understood that the fluid devices and methods described hereinare illustrative only and are not limiting upon the scope of theinvention, which is indicated by the following claims.

1-48. (canceled)
 49. A device comprising: at least three chambersarrayed in a plate, wherein (i) the device has a first face on one sideof the plate and a second face on a second, opposite side of the plate,(ii) each chamber, independent of any other chamber, has an inletopening to one face and an outlet opening to the other face; (iii) aplurality of the chambers are successively connected in series throughremovable conduits, wherein each conduit connects an outlet of onechamber with an inlet of another chamber; and (iv) the series ofchambers and conduits defines a fluid path connecting an inlet of afirst chamber through each of any intermediate chambers to an outlet ofa last chamber, wherein at least one of the chambers in the seriescontains a separation medium.
 50. The device of claim 49, wherein atleast some of the conduits pass through the plate and connect outletsopening to the second face with inlets opening to the first face. 51.The device of claim 50, wherein all of the conduits connect outletsopening to the second face with inlets opening to the first face. 52.The device of claim 50, wherein a plurality of the chambers in theseries are arrayed in a linear series, wherein each of the chambers isadjacent a channel that opens to both faces, and wherein the fluid pathbetween at least one outlet and inlet passes through the channels. 53.The device of claim 49, wherein at least some of the conduits connectoutlets opening to the first face with inlets opening to the first face,or outlets opening to the second face with inlets opening to the secondface.
 54. The device of claim 52, wherein at least one of the conduitsconnects an outlet opening to the second face with an inlet opening tothe second face, and wherein at least one of the conduits connects anoutlet opening to the first face with an inlet opening to the firstface.
 55. The device of claim 49, wherein the conduits are removablefrom the device.
 56. The device of claim 49, wherein the plurality ofchambers in the device is a multiple of
 8. 57. The device of claim 49,wherein the plurality of chambers in the device is a multiple of
 12. 58.The device of claim 49, wherein the chambers are arrayed in at least onelinear series.
 59. The device of claim 49, wherein the chambers arearrayed in a plurality of rows and columns.
 60. The device of claim 59,wherein the chambers are arrayed in an eight-by-twelve array.
 61. Thedevice of claim 49, comprising a plurality of series of chambers andconduits defining fluid paths.
 62. The device of claim 49, furthercomprising a collection plate comprising a plurality of wells that arearranged, in rows and columns, to correspond to the chambers of thedevice, wherein: each of the wells of the collection plate has an inlet;and upon disengagement of the conduits, the inlets of the wells of thecollection plate are configured to align with the chambers of thedevice.
 63. The device of claim 49, further comprising a pump that isconfigured to push or pull a fluid sample along the fluid path.
 64. Thedevice of claim 49, further comprising a drip-through microtiter platethat comprises wells corresponding to the chambers.
 65. A methodcomprising the steps of (a) providing a device comprising: at leastthree chambers arrayed in a plate, wherein (i) the device has a firstface on one side of the plate and a second face on a second, oppositeside of the plate, (ii) each chamber, independent of any other chamber,has an inlet opening to one face and an outlet opening to the otherface; (iii) a plurality of the chambers are successively connected inseries through removable conduits, wherein each conduit connects anoutlet of one chamber with an inlet of another chamber; (iv) the seriesof chambers and conduits defines a fluid path connecting an inlet of afirst chamber through each of any intermediate chambers to an outlet ofa last chamber; and (v) at least one chamber in the series comprises aseparation medium; (b) flowing a sample comprising a plurality ofanalytes along the fluid path from the inlet of a first chamber throughthe outlet of the last chamber, whereby the separation medium capturesanalytes having affinity for the medium; (c) removing the conduits thatconnect outlets to inlets from the device; (d) eluting captured analytesindependently from the chambers; and (e) collecting the eluted analytes.66. The method of claim 65, further comprising the step of detectinganalytes eluted from at least one chamber.
 67. The method of claim 65,wherein the step of detecting analytes is performed by mass spectrometryor gel electrophoresis.
 68. The method of claim 65, wherein the step ofeluting comprises eluting from at least one chamber in a plurality offractions.
 69. The method of claim 65, wherein the sample is selectedfrom the group consisting of blood, urine, cerebrospinal fluid andderivatives thereof.
 70. The method of claim 65, further comprising thestep of (b)(1) separating at least one of the analytes captured by theseparation medium into two or more mini-samples.
 71. The method of claim65, further comprising the step of (b)(1) separating at least one of theanalytes captured by the separation medium into two or more mini-samplesusing one or more of the following protocols: mass spectrometry,isoelectric focusing, hydrophobicity, and/or hydrophilicity.
 72. A kitcomprising: a device comprising: at least three chambers arrayed in aplate, wherein (i) the device has a first face on one side of the plateand a second face on a second, opposite side of the plate, (ii) eachchamber, independent of any other chamber, has an inlet opening to oneface and an outlet opening to the other face; (iii) a plurality of thechambers are successively connected in series through removableconduits, wherein each conduit connects an outlet of one chamber with aninlet of another chamber; and (iv) the series of chambers and conduitsdefines a fluid path connecting an inlet of a first chamber through eachof any intermediate chambers to an outlet of a last chamber; and atleast one container containing a separation medium.
 73. A devicecomprising: at least three chambers arrayed in a plate, wherein (i) thedevice has a first face on one side of the plate and a second face on asecond, opposite side of the plate, (ii) each chamber, independent ofany other chamber, has an inlet opening to one face and an outletopening to the other face; (iii) a plurality of the chambers aresuccessively connected in series through removable conduits, whereineach conduit connects an outlet of one chamber with an inlet of anotherchamber; and (iv) the series of chambers and conduits defines a fluidpath connecting an inlet of a first chamber through each of anyintermediate chambers to an outlet of a last chamber, wherein at leastone of the chambers in the series contains a separation medium and atleast one of chambers in the series does not contain a separationmedium.
 74. The device of claim 49, wherein at least one of the chambersin the series contains a separation medium that attracts and reversiblyretains one or more biomelecular components in a sample solution. 75.The device of claim 49, wherein some chambers in the series contain afirst separation medium and some other chambers in the series contain asecond separation medium.
 76. The device of claim 75, wherein the firstseparation medium is different from the second separation medium. 77.The device of claim 49, wherein each chamber in the series contains adifferent separation medium.
 78. The method of claim 65, wherein somechambers in the series contain a first separation medium and some otherchambers in the series contain a second separation medium that isdifferent from the first separation medium, and the separation mediacapture analytes having affinity for the media.